Engineered immunostimulatory bacterial strains and uses thereof

ABSTRACT

Provided are delivery immunostimulatory bacteria that have enhanced colonization of tumors, the tumor microenvironment and/or tumor-resident immune cells, and enhanced anti-tumor activity. The immunostimulatory bacteria are modified by deletion of genes encoding the flagella or modification of the genes so that functional flagella are not produced, and/or are modified by deletion of pagP or modification of pagP to produce inactive PagP product. As a result, the immunostimulatory bacteria are flagellin −  and/or pagP − . The immunostimulatory bacteria optionally have additional genomic modifications so that the bacteria are adenosine or purine auxotrophs. The bacteria optionally are one or more of asd − , purI −  and msbB − . The immunostimulatory bacteria, such as  Salmonella  species, are modified to encode immunostimulatory proteins that confer anti-tumor activity in the tumor microenvironment, and/or are modified so that the bacteria preferentially infect immune cells in the tumor microenvironment or tumor-resident immune cells and/or induce less cell death in immune cells than in other cells. Also provided are methods of inhibiting the growth or reducing the volume of a solid tumor by administering the immunostimulatory bacteria.

RELATED APPLICATIONS

This application is a continuation of International PCT Application No.PCT/US2019/041489, filed on Jul. 11, 2019, to Applicant ActymTherapeutics, Inc., inventors Christopher D. Thanos, Laura Hix Glickman,Justin Skoble and Alexandre Charles Michel lannello, and entitled“Engineered Immunostimulatory Bacterial Strains and Uses Thereof.”International PCT Application No. PCT/US2019/041489 claims the benefitof priority to U.S. Provisional Application Ser. No. 62/789,983, filedon Jan. 8, 2019, to Applicant Actym Therapeutics, Inc., inventorsChristopher D. Thanos, Laura Hix Glickman, Justin Skoble and AlexandreCharles Michel lannello, and entitled “Engineered ImmunostimulatoryBacterial Strains and Uses Thereof,” and to U.S. Provisional ApplicationSer. No. 62/828,990, filed on Apr. 3, 2019, to Applicant ActymTherapeutics, Inc., inventors Christopher D. Thanos, Laura Hix Glickman,Justin Skoble and Alexandre Charles Michel lannello, and entitled“Salmonella Strains Engineered To Colonize Tumors And The TumorMicroenvironment.”

Benefit of priority also is claimed to U.S. Provisional Application Ser.No. 62/789,983, filed on Jan. 8, 2019, to Applicant Actym Therapeutics,Inc., inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skobleand Alexandre Charles Michel lannello, and entitled “EngineeredImmunostimulatory Bacterial Strains and Uses Thereof.”

Benefit of priority also is claimed to U.S. Provisional Application Ser.No. 62/828,990, filed on Apr. 3, 2019, to Applicant Actym Therapeutics,Inc., inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skobleand Alexandre Charles Michel lannello, and entitled “Salmonella StrainsEngineered To Colonize Tumors And The Tumor Microenvironment.”

The immunostimulatory bacteria provided in each of these applicationscan be modified as described in this application and such bacteria areincorporated by reference herein. The subject matter of each of theseapplications is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Jul. 17, 2019, is 457 kilobytes in size,and is titled 1704SEQ001.txt.

FIELD OF THE INVENTION

Provided are immunostimulatory bacteria with genomes that are modifiedto, for example, increase colonization of the tumor microenvironment,tumors, and/or tumor-resident immune cells. This increase incolonization improves delivery of encoded therapeutic products andpermits systemic administration of the immunostimulatory bacteria.

BACKGROUND

The field of cancer immunotherapy has made great strides, as evidencedby the clinical successes of anti-CTLA4, anti-PD-1 and anti-PD-L1 immunecheckpoint antibodies (see, e.g., Buchbinder et al. (2015) J. Clin.Invest. 125: 3377-3383; Hodi et al. (2010) N. Engl. J. Med.363(8):711-723; and Chen et al. (2015) J. Clin. Invest. 125:3384-3391).Tumors have evolved a profoundly immunosuppressive environment. Theyinitiate numerous mechanisms to evade immune surveillance, reprogramanti-tumor immune cells to suppress immunity, and continually mutateresistance to the latest cancer therapies (see, e.g., Mahoney et al.(2015) Nat. Rev. Drug Discov. 14(8):561-584). Designing immunotherapiesthat overcome immune tolerance and escape, while limiting theautoimmune-related toxicities of current immunotherapies, challenges thefield of immuno-oncology. Hence, additional and innovativeimmunotherapies and other therapies are needed.

SUMMARY

Provided are bacteria modified to be immunostimulatory for anti-cancertherapy. Immunostimulatory bacteria, as provided herein, provide amulti-faceted approach to anti-tumor therapy. Bacteria provide aplatform in which there are numerous avenues for eliciting anti-tumorimmunostimulatory activity. As provided herein, bacteria, such asspecies of Salmonella, are fine-tuned to have potent anti-tumor activityby increasing their ability to accumulate in or target tumors,tumor-resident-immune cells, and/or the tumor microenvironment (TME).This is achieved by modifications that, for example, alter the type ofcells that they can infect (tropism), their toxicity, their ability toescape the immune system, such as complement, and/or the environments inwhich they can replicate. The immunostimulatory bacteria also canencode, for example, products that enhance or invoke an immune response,and therapeutic products. The immunostimulatory bacteria providedherein, by virtue of their improved colonization of tumors, the tumormicroenvironment, and/or tumor-resident immune cells, and theirresistance to complement and other anti-bacterial immune responses, canbe administered systemically.

The genomes of the bacteria provided herein are modified to increaseaccumulation in tumors and in tumor-resident immune cells, and also inthe tumor microenvironment. This is effected herein by deleting ordisabling genes responsible for infection or invasion of non-tumorcells, such as epithelial cells, and/or decreasing the cytopathogenicityof the bacteria, particularly to immune cells and tumor-resident immunecells.

Bacteria by their nature stimulate the immune system; bacterialinfection induces immune and inflammatory pathways and responses, someof which are desirable for anti-tumor treatment, and others, areundesirable. Modification of the bacteria by deleting or modifying genesand products that result in undesirable inflammatory responses, andadding or modifying genes and products that induce desirableimmunostimulatory anti-tumor responses, improves the anti-tumor activityof the bacteria.

Bacteria accumulate in tumor cells and tissues, and by replicatingtherein, can lyse cells. Bacteria migrate from the sites ofadministration and can accumulate in other tumors and tumor cells toprovide an abscopal effect. The bacteria provided herein are modified sothat they preferentially infect and accumulate in tumor-resident immunecells, tumors, and the tumor microenvironment.

Herein, all of these properties of bacteria are exploited to producedemonstrably immunostimulatory bacteria with a plurality of anti-tumoractivities and properties that can act individually and synergistically.

Provided are compositions, uses thereof and methods that modulate immuneresponses for the treatment of diseases, including for the treatment ofcancer. The compositions contain immunostimulatory bacteria providedherein. Methods of treatment and uses of the bacteria for treatment alsoare provided. The subjects for treatment include humans and otherprimates, pets, such as dogs and cats, and other animals, such ashorses.

Provided are pharmaceutical compositions containing theimmunostimulatory bacteria, and methods and uses thereof for treatmentof diseases and disorders, particularly proliferative disorders, such astumors, including solid tumors and hematologic malignancies.

Also provided are methods of inhibiting the growth or reducing thevolume of a solid tumor by administering the immunostimulatory bacteriaor pharmaceutical compositions, or using the compositions for treatment.For example, provided are methods of administering or using acomposition that contains, for a single dosage, an effective amount ofan attenuated Salmonella species to a subject, such as a human patient,having a solid tumor cancer. It is understood that all modifications tothe genome of the bacteria, such as anti-tumor therapeutics, and othermodifications of the bacterial genome and the plasmids described, can becombined in any desired combination.

Provided are immunostimulatory bacteria that have enhanced colonizationof tumors, the tumor microenvironment and/or tumor-resident immunecells, and enhanced anti-tumor activity. The immunostimulatory bacteriaare modified by deletion of genes encoding the flagella, or modificationof the genes so that functional flagella are not produced, and/ordeletion of pagP or modification of pagP to produce inactive PagPproduct. As a result, the immunostimulatory bacteria are flagellin⁻(fliC⁻/fljB⁻) and/or pagP⁻. Alternatively, or additionally, theimmunostimulatory bacteria can be pagP⁻/msbB⁻.

The immunostimulatory bacteria can be flagellin deficient, such as bydeletion of, or disruption in, a gene(s) encoding the flagella. Forexample, provided are immunostimulatory bacteria that contain deletionsin the genes encoding one or both of flagellin subunits fliC and fljB,whereby the bacterium is flagella deficient, and wherein the wild-typebacterium expresses flagella. The immunostimulatory bacteria also canhave a deletion or modification in the gene encoding endonuclease I(endA), whereby endA activity is inhibited or eliminated.

The immunostimulatory bacteria optionally have additional genomicmodifications so that the bacteria are adenosine or purine auxotrophs.The bacteria optionally are one or more of asd⁻, purI⁻ and msbB⁻. Theimmunostimulatory bacteria, such as Salmonella species, are modified toencode immunostimulatory proteins that confer anti-tumor activity in thetumor microenvironment, and/or are modified so that the bacteriapreferentially infect immune cells in the tumor microenvironment ortumor-resident immune cells, and/or induce less cell death in immunecells than in other cells. Also provided are methods of inhibiting thegrowth or reducing the volume of a solid tumor by administering theimmunostimulatory bacteria.

Provided are methods of increasing tumor colonization of animmunostimulatory bacterium, such as a Salmonella species, by modifyingthe genome of the immunostimulatory bacterium to be flagellin⁻(fliC⁻/fljB⁻) and/or pagP⁻.

The bacteria also contain plasmids that encode therapeutic products,such as anti-tumor agents, proteins that increase the immune response ofa subject, and inhibitory RNA (RNAi) that target immune checkpoints. Forexample, the plasmids can encode immunostimulatory proteins, such ascytokines, chemokines, and co-stimulatory molecules, that increase theanti-tumor response in the subject. The bacteria contain plasmids thatencode anti-cancer therapeutics, such as RNA, including microRNA, shRNA,and siRNA, and antibodies and antigen-binding fragments thereof that aredesigned to suppress, inhibit, disrupt or otherwise silence immunecheckpoint genes and products, and other targets that play a role inpathways that are immunosuppressive. The bacteria also can encode tumorantigens and tumor neoantigens on the plasmids to stimulate the immuneresponse against the tumors. The encoded proteins are expressed underthe control of promoters recognized by eukaryotic, such as mammalian andanimal, or viral, promoters.

Provided are immunostimulatory bacteria that contain a plasmid encodinga therapeutic product, such as an anti-cancer therapeutic; the genome ofthe immunostimulatory bacterium is modified so that it preferentiallyinfects tumor-resident immune cells and/or so that it induces less celldeath in tumor-resident immune cells.

Provided are immunostimulatory bacteria containing a plasmid encoding aproduct, generally a therapeutic product, such as an anti-cancertherapeutic product, under control of a eukaryotic promoter, where thegenome of the immunostimulatory bacterium is modified whereby thebacterium is flagellin⁻ (fliC⁻/fljB⁻) and/or pagP⁻, and whereby thewild-type bacteria have flagella. The bacteria can be one or both offlagellin⁻ (fliC⁻/fljB⁻) and pagP⁻. These immunostimulatory bacteriaexhibit increased colonization of tumors, the tumor microenvironmentand/or tumor-resident immune cells, and have increased anti-tumoractivity.

Among these immunostimulatory bacteria are those that are flagellin⁻(fliC⁻/fljB⁻), and whereby the therapeutic product is an anti-cancerproduct. In some embodiments, the bacteria are flagellin⁻ (fliC⁻/fljB⁻),and the product is an anti-cancer therapeutic protein or nucleic acid.

Among these immunostimulatory bacteria are those in which thetherapeutic product is a TGF-beta antagonist polypeptide, where thegenome of the immunostimulatory bacterium is modified so that thebacterium preferentially infects tumor-resident immune cells, and/or thegenome of the immunostimulatory bacterium is modified so that it inducesless cell death in tumor-resident immune cells (decreases pyroptosis),whereby the immunostimulatory bacterium accumulates in tumors or in thetumor microenvironment or in tumor-resident immune cells to therebydeliver the TGF-beta antagonist polypeptide to the tumormicroenvironment. The TGF-beta antagonist can be selected from among ananti-TGF-beta antibody, an anti-TGF-beta receptor antibody, and asoluble TGF-beta antagonist polypeptide. The nucleic acid encoding theTGF-beta antagonist polypeptide can include nucleic acid encoding asignal sequence for secretion of the encoded polypeptide, so that it isreleased into the tumor cells, tumor-resident immune cells, and/or thetumor microenvironment.

In other embodiments of any of the immunostimulatory bacteria providedherein, the plasmid encodes an immunostimulatory protein that confers,enhances, or contributes to an anti-tumor immune response in the tumormicroenvironment. Exemplary of immunostimulatory proteins that confer orcontribute to anti-tumor immunity in the tumor microenvironment is/areone or more of: IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-36gamma, IL-2 that has attenuated binding to IL-2Ra, IL-15/IL-15R alphachain complex, IL-18, IL-21, IL-23, IL-36γ, IL-2 modified so that itdoes not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α,interferon-β, interferon-γ, CCL3, CCL4, CCL5, proteins that are involvedin or that effect or potentiate recruitment/persistence of T cells,CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BBligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists,TGF-beta polypeptide antagonists, and members of the tumor necrosisfactor receptor (TNFR) superfamily.

In other embodiments of the immunostimulatory bacteria provided herein,the therapeutic product is an antibody or antigen-binding fragmentthereof. Exemplary of such is a Fab, Fab′, F(ab′)₂, single-chain Fv(scFv), Fv, disulfide-stabilized Fv (dsFv), nanobody, diabody fragment,or a single-chain antibody. The antibody or antigen-binding fragmentthereof can be humanized or human. Exemplary of an antibody orantigen-binding fragment thereof is an antagonist of PD-1, PD-L1,CTLA-4, VEGF, VEGFR2, or IL-6.

The immunostimulatory bacteria provided herein, including thosedescribed above, can contain a plasmid encoding a therapeutic productunder control of a eukaryotic promoter; the genome of theimmunostimulatory bacterium is modified whereby the bacterium ispagP⁻/msbB⁻, and optionally flagellin⁻ (fliC⁻/fljB⁻).

Exemplary of immunostimulatory bacteria are those that contain a plasmidencoding an immunostimulatory protein, where: an immunostimulatoryprotein, when expressed in a mammalian subject, confers or contributesto anti-tumor immunity in the tumor microenvironment; theimmunostimulatory protein is encoded on a plasmid in the bacterium undercontrol of a eukaryotic promoter; and the genome of theimmunostimulatory bacterium is modified so that it preferentiallyinfects tumor-resident immune cells. In other embodiments, theimmunostimulatory bacteria contain a sequence of nucleotides encoding animmunostimulatory protein, where the immunostimulatory protein, whenexpressed in a mammalian subject, confers or contributes to anti-tumorimmunity in the tumor microenvironment; the immunostimulatory protein isencoded on a plasmid in the bacterium under control of a eukaryoticpromoter; and the genome of the immunostimulatory bacterium is modifiedso that it induces less cell death in tumor-resident immune cells.Exemplary immunostimulatory proteins include cytokines and chemokines,and other immune stimulatory proteins, such as, for example one or moreof: IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-36 gamma, IL-2that has attenuated binding to IL-2Ra, IL-15/IL-15R alpha chain complex,IL-18, IL-21, IL-23, IL-36γ, IL-2 modified so that it does not bind toIL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ,CCL3, CCL4, CCL5, proteins that are involved in or that effect orpotentiate recruitment/persistence of T cells, CD40, CD40 ligand, CD28,OX40, OX40 ligand, 4-1BB, 4-1BB ligand, members of the B7-CD28 family,CD47 antagonists, TGF-beta polypeptide antagonists, and members of thetumor necrosis factor receptor (TNFR) superfamily.

These immunostimulatory bacteria can include modification(s) in thegenomes of the immunostimulatory bacteria so that the bacteria exhibitone or both of preferentially infecting tumor-resident immune cells, andinducing less cell death in tumor-resident immune cells. Theimmunostimulatory bacteria can also include a mutation in the genomethat reduces toxicity or infectivity of non-immune cells in a host.

Modifications of the bacterial genome include pagP⁻, or pagP⁻ andflagellin⁻ (fliC⁻/fljB⁻). In other embodiments, the immunostimulatorybacteria is one or more of purI⁻ (purM⁻), msbB⁻, purD⁻, flagellin⁻(fliC⁻/fljB⁻), pagP⁻, adrA⁻, csgD⁻, qseC⁻, and hilA⁻, such as flagellin⁻(fliC⁻/fljB⁻)/pagP⁻/msbB⁻/purI⁻, or flagellin⁻(fliC⁻/fljB⁻)/pagP⁻/msbB⁻/purI⁻/hilA⁻. In other embodiments, theimmunostimulatory bacteria are hilA⁻ and/or flagellin⁻ (fliC⁻/fljB⁻) orpagP⁻ or pagP⁻/msbB⁻ or the immunostimulatory bacteria are hilA⁻ or theimmunostimulatory bacteria are flagellin⁻ (fliC⁻/fljB⁻) and pagP⁻. Thegenome modifications, among other properties, can increase targeting toor colonization of the tumor microenvironment and/or tumor-residentimmune cells, and/or render the bacteria substantially or completelyresistant to inactivation by complement. These properties improve theuse of the bacteria as therapeutics, and permit systemic administration.

In the immunostimulatory bacteria provided herein, the nucleic acidencoding the therapeutic product is operatively linked for expression toa nucleic acid encoding a secretory signal, whereby, upon expression ina host, the immunostimulatory protein is secreted. The therapeuticproduct can be a protein, such as an immunostimulatory protein, or anucleic acid, such as a CRISPR cassette or an RNAi.

In all embodiments, the immunostimulatory bacteria can be auxotrophicfor adenosine, or for adenosine and adenine. The immunostimulatorybacteria provided herein can include modifications in the genome wherebythe bacterium preferentially infects tumor-resident immune cells, and/orthe genome of the immunostimulatory bacterium is modified so that itinduces less cell death in tumor-resident immune cells (decreasespyroptosis), whereby the immunostimulatory bacterium accumulates intumors or in the tumor microenvironment or in tumor-resident immunecells to thereby deliver an encoded therapeutic product.

In the immunostimulatory bacteria, the plasmid encodes the therapeuticproduct under control of a eukaryotic promoter so that it is expressedin a eukaryotic host, such as a human or other mammal. The therapeuticproduct generally is an anti-cancer therapeutic, such as an anti-cancertherapeutic protein that stimulates the immune system of the host. Othertherapeutic products include antibodies and antigen-binding fragmentsthereof, and nucleic acids, such as RNAi. These products can be designedto inhibit, suppress, or disrupt a target, such as an immune checkpointand other such targets that impair the ability of the immune system of asubject to recognize the tumor cells.

The unmodified immunostimulatory bacteria can be a wild-type strain oran attenuated strain. The genome modifications provided and describedherein attenuate the bacteria outside of the tumor microenvironment ortumors; the modifications, among other properties, alter the infectivityof the bacteria. Exemplary of bacteria that can be modified as describedherein is Salmonella, such as a Salmonella typhimurium strain. Exemplaryof Salmonella typhimurium strains are attenuated and wild-type strains,such as, for example, Salmonella typhimurium strains derived fromstrains designated as AST-100, VNP20009, YS1646 (ATCC #202165), RE88,SL7207, χ 8429, χ 8431, χ 8468, or a wild-type strain with ATCCaccession no. 14028.

As discussed above, provided are immunostimulatory bacteria containing aplasmid encoding a product under control of a eukaryotic promoter, wherethe genome of the immunostimulatory bacterium is modified whereby thebacterium is pagP⁻/msbB⁻. Deletion of msbB alters the acyl compositionof the lipid A domain of lipopolysaccharide (LPS), the major componentof the outer membranes of Gram-negative bacteria, such that the bacteriapredominantly produce penta-acylated LPS instead of the more toxic andpro-inflammatory hexa-acylated LPS. In wild type S. typhimurium,expression of pagP results in hepta-acylated lipid A, while in an msbB⁻mutant, the induction of pagP results in hexa-acylated LPS. Thus, apagP⁻/msbB⁻ mutant produces only penta-acylated LPS, resulting in lowerinduction of pro-inflammatory cytokines, and enhanced tolerability,which allows for higher dosing in humans. Higher dosing leads toincreased colonization of tumors, tumor-resident immune cells, and thetumor microenvironment. Because of the resulting change in bacterialmembranes and structure, the host immune response, such as complementactivity, is altered so that the bacteria are not eliminated uponsystemic administration. For example, it is shown herein that pagP/msbB⁻mutant strains have increased resistance to complement inactivation andenhanced stability in human serum. These bacteria also can be flagellin⁻(fliC⁻/fljB⁻), which further enhances tolerability, resistance tocomplement inactivation, and tumor/TME/tumor-resident immune cellcolonization. The bacteria also can comprise other modifications asdescribed herein, including modifications that alter the cells that theycan infect, resulting in accumulation in the tumor microenvironment,tumors and tumor-resident immune cells. Hence, the immunostimulatorybacteria provided herein can be systemically administered and exhibit ahigh level of tumor, tumor microenvironment and/or tumor-resident immunecell colonization. The immunostimulatory bacteria can be purI⁻ (purM⁻),and one or more of asdt, msbB⁻, and one or both of flagellin⁻(fliC⁻/fljB⁻) and pagP⁻.

The immunostimulatory bacteria can be aspartate-semialdehydedehydrogenase⁻ (asd⁻), such as by virtue of disruption or deletion ofall or a portion of the endogenous gene encoding aspartate-semialdehydedehydrogenase (asd), whereby endogenous asd is not expressed. Theseimmunostimulatory bacteria can be modified to encodeaspartate-semialdehyde dehydrogenase (asd) on a plasmid under control ofa bacterial promoter so that the bacteria can be produced in vitro.

The immunostimulatory bacteria can be rendered auxotrophic forparticular nutrients that are rich or that accumulate in the tumormicroenvironment, such as adenosine and adenine. Also, they can bemodified to be auxotrophic for such nutrients to reduce or eliminatetheir ability to replicate. The inactivated/deleted bacterial genomegenes can be complemented by providing them on a plasmid under thecontrol of promoters recognized by the host.

The products encoded on the plasmids for expression in a eukaryotic,such as a human, host, are under control of eukaryotic regulatorysequences, including eukaryotic promoters, such as promoters recognizedby RNA polymerase II or III. These include mammalian RNA polymerase IIpromoters. Viral promoters also can be used. Exemplary viral promoters,include, but are not limited to, a cytomegalovirus (CMV) promoter, anSV40 promoter, an Epstein Barr virus (EBV) promoter, a herpes viruspromoter, and an adenovirus promoter. Other RNA polymerase II promotersinclude, but are not limited to, an elongation factor-1 (EF1) alphapromoter, a UbC promoter (lentivirus), a PGK (3-phosphoglycerate kinase)promoter, and a synthetic promoter such as a CAGG (or CAG) promoter. Thesynthetic CAG promoter contains the cytomegalovirus (CMV) early enhancerelement (C); the promoter, the first exon and the first intron ofchicken beta-actin gene (A); and the splice acceptor of the rabbitbeta-globin gene (G). Other strong regulatable or constitutive promoterscan be used. The regulatory sequences also include terminators,enhancers, and secretory and other trafficking signals.

The plasmids included in the immunostimulatory bacteria can be presentin low copy number or medium copy number, such as by selection of anorigin of replication that results in medium-to-low copy number, such asa low copy number origin of replication. It is shown herein that theanti-tumor activity and other properties of the bacteria are improvedwhen the plasmid is present in low to medium copy number, where mediumcopy number is less than 150 or less than about 150 and more than 20 orabout 20 or is between 20 or 25 and 150 copies, and low copy number isless than 25 or less than 20 or less than about 25 or less than about 20copies.

These immunostimulatory bacteria can be modified so that the bacteriapreferentially infect tumor-resident immune cells, and/or the genome ofthe immunostimulatory bacteria can be modified so that they induces lesscell death in tumor-resident immune cells (decrease pyroptosis), wherebythe immunostimulatory bacteria accumulate in tumors or in the tumormicroenvironment or in tumor-resident immune cells.

As discussed above, the genome of the immunostimulatory bacteria also ismodified so that the bacteria preferentially infect immune cells, suchas tumor-resident immune cells, such as myeloid cells, such as cellsthat are CD45⁺, and/or the genome is modified so that the bacteriainduce less cell death in tumor-resident immune cells (decreasedpyroptosis) than the unmodified bacteria. As a result, theimmunostimulatory bacteria accumulate, or accumulate to a greater extentthan those without the modifications, in tumors or in the tumormicroenvironment or in tumor-resident immune cells, to thereby deliverthe therapeutic product or products encoded on the plasmid. The bacteriacan be one or more of flagellin⁻ (fliC⁻/fljB⁻), pagP⁻, and msbB⁻, andcan include other such modifications as described herein. The bacteriacan be auxotrophic for adenosine, and/or purI⁻ (purM⁻) and/or asd⁻.

The immunostimulatory bacteria provided herein can include amodification of the bacterial genome, whereby the bacteria induce lesscell death in tumor-resident immune cells; and/or a modification of thebacterial genome, whereby the bacteria accumulate more effectively intumors, the tumor microenvironment, or tumor-resident immune cells, suchas tumor resident CD45⁺ cells, and myeloid cells.

For example, the immunostimulatory bacteria can include deletions ormodifications of one or more genes or operons involved in SPI-1 invasion(and/or SPI-2), whereby the immunostimulatory bacteria do not invade orinfect epithelial cells. Exemplary of genes that can be deleted orinactivated are one or more of avrA, hilA, hilD, invA, invB, invC, invE,invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS,orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC,sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP. Elimination of theability to infect epithelial cells also can be achieved by engineeringthe immunostimulatory bacteria herein to contain knockouts or deletionsof genes encoding proteins involved in SPI-1-independent invasion, suchas one or more of the genes selected from among rck, pagN, hlyE, pefI,srgD, srgA, srgB, and srgC. Similarly, the immunostimulatory bacteriacan include deletions in genes and/or operons in SPI-2, for example, toengineer the bacteria to escape the Salmonella-containing vacuole (SCV).These genes include, for example, sifA, sseJ, sseL, sopD2, pipB2, sseF,sseG, spvB, and steA.

The immunostimulatory bacteria provided herein also can contain asequence of nucleotides encoding an immunostimulatory protein that, whenexpressed in a mammalian subject, confers or contributes to anti-tumorimmunity in the tumor microenvironment; the immunostimulatory protein isencoded on a plasmid in the bacterium under control of a eukaryoticpromoter. Exemplary promoters include, but are not limited to, anelongation factor-1 (EF1) alpha promoter, or a UbC promoter, or a PGKpromoter, or a CAGG or CAG promoter.

Additionally, the genome of the immunostimulatory bacterium is modifiedso that it preferentially infects tumor-resident immune cells. This isachieved by deleting or disrupting bacterial genes that play a role ininvasiveness or infectivity of the bacteria, and/or that play a role ininducing cell death. The bacteria are modified to preferentially infecttumor-resident immune cells, and/or induce less cell death intumor-resident immune cells than in other cells that the bacteria caninfect, than unmodified bacteria.

The immunostimulatory bacteria also can encode a therapeutic product,such as inhibitory RNA (RNAi), immunostimulatory proteins such ascytokines, chemokines and co-stimulatory molecules, other proteins thatincrease the immune response in a subject, and other anti-tumor agents,that, when expressed in a mammalian subject, confer or contribute toanti-tumor immunity. The therapeutic product is encoded on a plasmid inthe bacterium under control of a eukaryotic promoter. The genome of theimmunostimulatory bacterium is modified so that it induces less celldeath in tumor-resident immune cells. The plasmid generally is presentin low or medium copy number.

Also provided are immunostimulatory bacteria that encode animmunostimulatory protein on a plasmid in the bacterium under control ofa eukaryotic promoter, that, when expressed in a mammalian subject,confers or contributes to anti-tumor immunity in the tumormicroenvironment. The immunostimulatory bacteria can be modified to havereduced pathogenicity, whereby infection of epithelial and/or othernon-immune cells is reduced, relative to the bacterium without themodification. These include modification of the type 3 secretion system(T3SS) or type 4 secretion system (T4SS), such as modification of theSPI-1 pathway of Salmonella as described and exemplified herein. Thebacteria further can be modified to induce less cell death, such as bydeletion or disruption of nucleic acid encoding lipid Apalmitoyltransferase (pagP), which reduces virulence of the bacteria.

The genome of the immunostimulatory bacteria provided herein can bemodified to increase or promote infection of immune cells, particularlyimmune cells in the tumor microenvironment, such as phagocytic cells.This includes reducing infection of non-immune cells, such as epithelialcells, or increasing infection of immune cells. The bacteria also can bemodified to decrease pyroptosis in immune cells. Numerous modificationsof the bacterial genome can do one or both of increasing infection ofimmune cells and decreasing pyroptosis. The immunostimulatory bacteriaprovided herein include such modifications, for example, deletionsand/or disruptions of genes involved in the SPI-1 T3SS pathway, such asdisruption or deletion of hilA, and/or disruption/deletion of genesencoding flagellin, rod protein (PrgJ), needle protein (PrgI) and QseC.

The immunostimulatory bacteria can be one or more of purI⁻ (purM⁻),msbB⁻, purD⁻, flagellin⁻ (fliC⁻/fljB⁻), pagP⁻, adrA⁻, csgD⁻, qseC⁻, andhilA⁻, and particularly flagellin⁻ (fliC⁻/fljB⁻) and/or pagP⁻, and/ormsbB⁻/pagP⁻. For example, the immunostimulatory bacteria can includemutations in the genome, such as gene deletions or disruptions thatreduce toxicity or infectivity of non-immune cells in a host. Forexample, the immunostimulatory bacteria can be pagP⁻. As anotherexample, the immunostimulatory bacteria can be hilA⁻ and/or flagellin⁻(fliC⁻/fljB⁻), and also can be pagP⁻. Thus, for example, theimmunostimulatory bacteria can encode an immunostimulatory protein, suchas a cytokine, and the bacteria can be modified so that they accumulateand express the cytokine in the tumor microenvironment (TME), therebydelivering an immunotherapeutic anti-tumor product into the environmentin which it has beneficial activity, and avoiding adverse or toxic sideeffects from expression in other cells/environments. The nucleic acidencoding the immunostimulatory protein can be operatively linked forexpression to nucleic acid encoding a secretory signal, whereby, uponexpression in a host, the immunostimulatory protein is secreted into thetumor microenvironment.

The immunostimulatory bacteria provided herein include any of thestrains and bacteria described in co-pending U.S. application Ser. No.16/033,187, or published International Application No. PCT/US2018/041713(published as WO 2019/014398), further modified to express animmunostimulatory protein and/or to preferentially infect and/or to beless toxic in immune cells in the tumor microenvironment or intumor-resident immune cells, as described and exemplified herein.

The immunostimulatory bacteria can be aspartate-semialdehydedehydrogenase⁻ (asd⁻), such as by virtue of disruption or deletion ofall or a portion of the endogenous gene encoding aspartate-semialdehydedehydrogenase (asd), whereby the endogenous asd is not expressed. Theimmunostimulatory bacteria can be modified to encodeaspartate-semialdehyde dehydrogenase (asd) on a plasmid under control ofa bacterial promoter for growing the bacteria in vitro, so that bacteriawill have limited replication in vivo.

The immunostimulatory bacteria provided herein can encode, on a plasmid,an immunostimulatory protein as a therapeutic product. Theimmunostimulatory protein can be a cytokine, such as a chemokine, or aco-stimulatory molecule. Exemplary of immunostimulatory proteins areIL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-15/IL-15R alphachain complex, IL-36 gamma, IL-18, CXCL9, CXCL10, CXCL11, CCL3, CCL4,CCL5, proteins that are involved in or that effect or potentiate therecruitment/persistence of T cells, CD40, CD40 Ligand (CD40L), OX40,OX40 Ligand (OX40L), 4-1BB, 4-1BB Ligand (4-1BBL), members of theB7-CD28 family, and members of the tumor necrosis factor receptor (TNFR)superfamily.

The immunostimulatory bacteria optionally can include a sequence ofnucleotides encoding inhibitory RNA (RNAi) that inhibits, suppresses ordisrupts expression of an immune checkpoint. The RNAi can be encoded ona plasmid in the bacterium. The nucleotides encoding theimmunostimulatory protein, and optionally an RNAi, can be on a plasmidpresent in low to medium copy number.

The immunostimulatory bacteria also can encode therapeutic products,such as RNAi or a CRISPR cassette that inhibits, suppresses or disruptsexpression of an immune checkpoint or other target whose inhibition,suppression or disruption increases the anti-tumor immune response in asubject; the RNAi or CRISPR cassette is encoded on a plasmid in thebacterium. Other therapeutic products include, for example, antibodiesthat bind to immune checkpoints to inhibit their activities.

RNAi includes all forms of double-stranded RNA that can be used tosilence the expression of targeted nucleic acids. RNAi includes shRNA,siRNA and microRNA (miRNA). Any of these forms can be interchanged inthe embodiments disclosed and described herein. In general, the RNAi isencoded on a plasmid in the bacterium. The plasmids can include otherheterologous nucleic acids that encode products of interest thatmodulate or add activities or products to the bacterium, or other suchproducts that can modulate the immune system of a subject to be treatedwith the bacterium. Bacterial genes also can be added, deleted ordisrupted. These genes can encode products for growth and replication ofthe bacteria, or products that also modulate the immune response of thehost to the bacteria.

The immunostimulatory bacteria provided herein also can be auxotrophicfor adenosine, or for adenosine and adenine.

Bacterial species for modification as described herein, carryingplasmids as described herein, include, but are not limited to, forexample, strains of Salmonella, Shigella, Listeria, E. coli, andBifidobacteriae. For example, species include Shigella sonnei, Shigellaflexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonellatyphi, Salmonella typhimurium, Salmonella gallinarum, and Salmonellaenteritidis.

Species include, for example, strains of Salmonella, Shigella, E. coli,Bifidobacteriae, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella,Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium,Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium,Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter,Bacillus, and Erysipelothrix, or an attenuated strain thereof or amodified strain thereof of any of the preceding list of bacterialstrains.

Other suitable bacterial species include Rickettsia, Klebsiella,Bordetella, Neisseria, Aeromonas, Franciesella, Corynebacterium,Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium,Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio,Bacillus, and Erysipelothrix. For example, Rickettsia Rikettsiae,Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri,Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis,Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida,Franciesella tularensis, Corynebacterium pseudotuberculosis, Citrobacterfreundii, Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus,Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi,Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillussubtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica,Rochalimaea quintana, and Agrobacterium tumerfacium.

Salmonella is exemplified herein, and particularly Salmonellatyphimurium strains. The Salmonella can be wild-type species orattenuated species. Exemplary of some attenuated species are the strainsdesignated YS1646 (ATCC #202165) or VNP20009. Other strains include,RE88, SL7207, χ 8429, χ 8431, and x 8468. Exemplary of wild-type orunattenuated species include, for example, the wild-type straindeposited as ATCC 14028, or a strain having all of the identifyingcharacteristics of ATCC 14028. The modifications herein attenuate anystrain by constraining the cells that the bacteria can infect or inwhich they can replicate.

These strains can be further modified to encode immunostimulatoryproteins and/or immune modulatory proteins. For example, theimmunostimulatory bacteria can encode immunostimulatory proteins, suchas cytokines, that increase the immune response in the tumormicroenvironment. The immunostimulatory bacteria also can be modified topreferentially infect immune cells in the tumor microenvironment or toinfect tumor-resident immune cells, and/or to induce less cell death insuch immune cells, as described herein. Sequences thereof anddescriptions are provided in the detailed description, examples andsequence listing. The immunostimulatory bacteria can be derived fromattenuated strains of bacteria, or they become attenuated by virtue ofthe modifications described herein, such as deletion of asd, wherebyreplication is limited in vivo.

It is understood that instances in which bacterial genes are modifiedand referenced herein, they are referenced with respect to theirdesignation (name) in Salmonella species, which is exemplary of bacteriafrom which immunostimulatory bacteria can be produced. The skilledperson recognizes that other species have corresponding proteins, butthat their designation or name can be different from the name inSalmonella. The generic disclosure herein, however, can be applied toother bacterial species. For example, as shown herein, deletion orinactivation of flagellin (fliC⁻/fljB⁻) in Salmonella and/or pagPresults in increased colonization of tumors. Similar genes encodingflagella, or similar functions for infection, can be modified in otherbacterial species to achieve increased tumor colonization. Similarly,inactivation/deletion of bacterial products, such as the products ofpagP and/or msbB, as described herein, can reduce complement activationand/or other inflammatory responses, thereby increasing targeting totumors, tumor-resident immune cells and the tumor microenvironment.Corresponding genes in other species that are involved in activating thecomplement pathway or other inflammatory pathway, can be deleted, asexemplified herein for Salmonella.

The immunostimulatory bacteria provided herein encode inhibitors ofvarious genes that reduce anti-tumor immune responses, and/or expressgenes and/or gene products that contribute to anti-tumor immuneresponses and/or products that stimulate the immune system, such asimmunostimulatory proteins, such as cytokines, chemokines, andco-stimulatory molecules, and thereby are immunostimulatory. Adenosineauxotrophy is immunostimulatory. Other therapeutic products that can beencoded on the plasmids are nucleic acids, such as inhibitory RNA(RNAi), such as shRNA or microRNA or siRNA, targeted for disruption orinhibition of expression of TREX1, PD-L1, VISTA (the gene encodingV-domain Ig suppressor of T cell activation), TGF-beta, and CTNNB1 (thegene that encodes β-catenin), among others, combinations thereof, andcombinations thereof with any RNAi's that inhibit, suppress or disruptexpression of other immune suppressive genes whose expression isactivated or enhanced by tumors or the tumor microenvironment (TME).Expression of these RNAs exploits two independent immunostimulatorypathways, and leads to enhanced tumor colonization in a single therapy.The effects of this combination are enhanced by the strains providedherein that are auxotrophic for adenosine, which provides preferentialaccumulation in, or recruitment into, adenosine-rich immunosuppressivetumor microenvironments. Reducing adenosine in such TMEs furtherenhances the immunostimulatory effects. Such combinations of traits inany of the bacterial strains known, or that can be engineered fortherapeutic administration, provide similar immunostimulatory effects.

Among the targets is TGF-beta, which has three isoforms: 1, 2 and 3.Among the targets is TGF-beta, particularly isoform 1, and not isoforms2 and 3. Toxicities are associated with inhibition of isoforms 2 and 3.For example, cardiac valve toxicity is associated with inhibition ofisoform 2. Isoform 1 is present in most cancers (see, e.g., TCGAdatabase). It is advantageous to inhibit only isoform 1. RNAi can beadvantageously employed for this purpose, since it can be designed tovery specifically recognize a target. For TGF-beta, specific inhibitionof isoform 1 can be effected by targeting a sequence unique to isoform 1that is not present in isoforms 2 or 3, or to select a sequence totarget isoforms 1 and 3, and not 2. Also provided are immunostimulatorybacteria in which the plasmid encodes an shRNA or microRNA thatspecifically inhibits, suppresses or disrupts expression of TGF-betaisoform 1 but not TGF-beta isoform 2 or TGF-beta isoform 3; or theplasmid encodes an shRNA or microRNA that specifically inhibits,suppresses or disrupts expression of TGF-beta isoforms 1 and 3, but notisoform 2.

RNAi, such a miRNA- or shRNA-mediated gene disruption of PD-L1 by theimmunostimulatory bacteria provided herein, also improves colonizationof tumors, the TME, and/or tumor-resident immune cells. It has beenshown that knockout of PD-L1 enhances S. typhimurium infection. Forexample, an at least 10-fold higher bacterial load in PD-L1 knockoutmice than in wild-type mice has been observed, indicating that PD-L1 isprotective against S. typhimurium infection (see, e.g., Lee et al.(2010) Immunol. 185:2442-2449).

Engineered immunostimulatory bacteria, such as the S. typhimuriumimmunostimulatory bacteria provided herein, contain multiple synergisticmodalities to induce immune re-activation of cold tumors to promotetumor antigen-specific immune responses, while inhibiting immunecheckpoint pathways that the tumor utilizes to subvert and evade durableanti-tumor immunity. Included in embodiments is adenosine auxotrophy andenhanced vascular disruption. This improvement in tumor targetingthrough adenosine auxotrophy and enhanced vascular disruption increasespotency, while localizing the inflammation to limit systemic cytokineexposure and the autoimmune toxicities observed with other immunotherapymodalities.

The heterologous therapeutic proteins and other products, such as theimmunostimulatory proteins, antibodies, and RNAs, are expressed onplasmids under the control of promoters that are recognized by theeukaryotic host cell transcription machinery, such as RNA polymerase II(RNAP II) and RNA polymerase III (RNAP III) promoters. RNAP IIIpromoters generally are constitutively expressed in a eukaryotic host;RNAP II promoters can be regulated. The therapeutic products are encodedon plasmids stably expressed by the bacteria. Exemplary of such bacteriaare Salmonella strains, generally attenuated strains, either attenuatedby passage or other methods, or by virtue of modifications describedherein, such as adenosine auxotrophy. Exemplary of Salmonella strainsare modified S. typhimurium strains that have a defective asd gene.These bacteria can be modified to include carrying a functional asd geneon the introduced plasmid; this maintains selection for the plasmid sothat an antibiotic-based plasmid maintenance/selection system is notneeded. The asd defective strains that do not contain a functional asdgene on a plasmid are autolytic in the host.

The promoters can be selected for the environment of the tumor cell,such as a promoter expressed in a tumor microenvironment (TME), apromoter expressed in hypoxic conditions, or a promoter expressed inconditions where the pH is less than 7.

Plasmids can be present in many copies or fewer. This can be controlledby selection of elements, such as the origin of replication. Low, mediumand high copy number plasmids and origins of replication are well knownto those of skill in the art and can be selected. In embodiments of theimmunostimulatory bacteria herein, the plasmid can be present in low tomedium copy number, such as about 150 or 150 and fewer copies, to lowcopy number, which is less than about 25 or about 20 or 25 copies.Exemplary origins of replication are those derived from pBR322, p15A,pSC101, pMB1, colE1, colE2, pPS10, R6K, R1, RK2, and pUC.

The plasmids can include RNAi such that the RNA inhibits, suppresses ordisrupts expression of an immune checkpoint or other target and,additionally, their products. The plasmids also can include sequences ofnucleic acids encoding a listeriolysin O (LLO) protein lacking thesignal sequence (cytoLLO), a CpG motif, a DNA nuclear targeting sequence(DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element. Theimmunostimulatory bacterium that comprises nucleic acid can include aCpG motif recognized by toll-like receptor 9 (TLR9). The CpG motif canbe encoded on the plasmid. The CpG motif can be included in, or is partof, a bacterial gene that is encoded on the plasmid. For example, thegene that comprises CpGs can be asd, encoded on the plasmid. Theimmunostimulatory bacteria provided herein can include one or more of aCpG motif, an asd gene selectable marker for plasmid maintenance, and aDNA nuclear targeting sequence.

The immunostimulatory bacteria provided herein can encode two or moredifferent RNA molecules that inhibit, suppress or disrupt expression ofan immune checkpoint and/or an RNA molecule that encodes an inhibitor ofa metabolite that is immunosuppressive or is in an immunosuppressivepathway.

The immunostimulatory bacteria provided herein can beaspartate-semialdehyde dehydrogenase⁻ (asd⁻), which permits growth indiaminopimelic acid (DAP) supplemented medium, but limits replication invivo when administered to subjects for treatment. Such bacteria will beself-limiting, which can be advantageous for treatment. The bacteriumcan be asd⁻ by virtue of disruption or deletion of all or a portion ofthe endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd),whereby the endogenous asd is not expressed. In other embodiments, thegene encoding aspartate-semialdehyde dehydrogenase can be included onthe plasmid for expression in vivo.

Any of the immunostimulatory bacteria provided herein can includenucleic acid, generally on the plasmid, that includes a CpG motif or aCpG island, wherein the CpG motif is recognized by toll-like receptor 9(TLR9). Nucleic acid encoding CpG motifs or islands are plentiful inprokaryotes, and, thus, the CpG motif can be included in, or can be apart of, a bacterial gene that is encoded on the plasmid. For example,the bacterial gene asd contains immunostimulatory CpGs.

The immunostimulatory bacteria provided herein can be auxotrophic foradenosine, or adenosine and adenine. Any of the bacteria herein can berendered autotrophic for adenosine, which advantageously can increasethe anti-tumor activity, since adenosine accumulates in many tumors, andis immunosuppressive.

The immunostimulatory bacteria provided herein can be flagellindeficient, where the wild-type bacterium comprises flagella. They can berendered flagellin deficient by disrupting or deleting all or a part ofthe gene or genes that encode the flagella. For example, provided areimmunostimulatory bacteria that have deletions in the genes encoding oneor both of flagellin subunits fliC and fljB, whereby the bacteria isflagella deficient.

The immunostimulatory bacteria provided herein can include nucleic acidencoding cytoLLO, which is a listeriolysin O (LLO) protein lacking theperiplasmic secretion signal sequence, so that it accumulates in thecytoplasm. This mutation is advantageously combined with asdc bacteria.LLO is a cholesterol-dependent pore forming hemolysin from Listeriamonocytogenes that mediates phagosomal escape of bacteria. When theautolytic strain is introduced into tumor-bearing hosts, such as humans,the bacteria are taken up by phagocytic immune cells and enter thevacuole. In this environment, the lack of DAP prevents bacterialreplication, and results in autolysis of the bacteria in the vacuole.Lysis then releases the plasmid and the accumulated LLO forms pores inthe cholesterol-containing vacuole membrane and allows for delivery ofthe plasmid into the cytosol of the host cell.

The immunostimulatory bacteria can include a DNA nuclear targetingsequence (DTS), such as an SV40 DTS, encoded on the plasmid.

The immunostimulatory bacteria can have a deletion or modification inthe gene encoding endonuclease-1 (endA), whereby endA activity isinhibited or eliminated. Exemplary of these are immunostimulatorybacteria that contain one or more of a CpG motif, an asd gene selectablemarker for plasmid maintenance and a DNA nuclear targeting sequence.

The immunostimulatory bacteria can contain nucleic acids on the plasmidencoding two or more different RNA molecules that inhibit, suppress ordisrupt expression of an immune checkpoint or an RNA molecule thatencodes an inhibitor of a metabolite that is immunosuppressive or is inan immunosuppressive pathway.

The nucleic acids encoding the RNAi, such as shRNA or miRNA or siRNA caninclude a transcriptional terminator following the RNA-encoding nucleicacid. In all embodiments, the RNAi encoded on the plasmid in theimmunostimulatory bacteria can be short hairpin RNAs (shRNAs) ormicro-RNAs (miRNAs).

The immunostimulatory bacteria can additionally encode a therapeuticproduct, such as RNAi that inhibits, suppresses, disrupts or silencesexpression of immune checkpoints and other targets whose inhibition,suppression, disruption or silencing is immunostimulatory, or anantibody or other binding protein that inhibits expression of thesetargets. These targets include, but are not limited to, one or more ofthree prime repair exonuclease 1 (TREX1), PD-1, PD-L1 (B7-H1), VEGF,TGF-beta isoform 1, beta-catenin, CTLA-4, PD-L2, PD-2, IDO1, IDO2,SIRPa, CD47, VISTA (B7-H5), LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT,Galectin-9, CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, B7-H3,ICOS, GITR, B7-H4, B7-H6, CD27, CD40, CD40L, CD48, CD70, CD80, CD86,CD137 (4-1BB), CD200, CD272 (BTLA), CD160, CD39, CD73, A2a receptor, A2breceptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2/4, OX40,OX-40L, KIR, TIM1, TIM4, STAT3, Stabilin-1 (CLEVER-1), DNase II andRNase H2. For example, any of the immunostimulatory bacteria can containRNA that inhibits, suppresses or disrupts expression of one or acombination of TREX1, PD-L1, VISTA, TGF-beta, such as TGF-beta isoform 1or isoforms 1 and 3, beta-catenin, SIRP-alpha, VEGF, RNase H2, DNase II,and CLEVER-1/Stabilin-1. Cluster of Differentiation 47 (CD47), alsoknown as integrin associated protein (IAP), is a transmembrane receptorbelonging to the immunoglobulin superfamily of proteins. CD47 isubiquitously expressed on cells and serves as a marker forself-recognition, preventing phagocytosis. CD47 mediates its effectsthrough interactions with several other proteins, includingthrombospondin (TSP) and signal regulatory protein-alpha (SIRPa). Theinteraction between SIRPa on phagocytic cells and CD47 on target cellshelps ensure that target cells do not become engulfed by the phagocyticcells. Certain cancers co-opt the CD47-based immune evasion mechanism ofa cell by increasing expression of CD47 on the cell surface of thecancer cell, thus avoiding clearance by the immune system. TargetingCD47-expressing cells in a subject results in toxicities. Encoding aCD47 inhibitory molecule, such as an antibody or antibody fragment, suchas a nanobody (see, e.g., Sockolosky et al. (2016) Proc. Natl. Acad.Sci. U.S.A. 113:E2646-E2654) on plasmids in the immunostimulatorybacteria provided herein results in expression of the anti-CD47 productin the tumor microenvironment or tumor. Anti-CD47 antibody fragmentshave been encoded in bacteria, such as E. coli that are administeredintratumorally (see, e.g., Chowdhury et al. (2019) Nature Medicine25:1057-1063). The bacteria herein have improved targeting andcolonization of the tumor microenvironment, tumors, and/ortumor-resident immune cells, and, thus, can more effectively deliver theanti-CD47 antibody or antibody fragment. The immunostimulatory bacteriaprovided herein can be systemically administered to colonize tumors andthe tumor microenvironment.

Provided are immunostimulatory bacteria where the plasmid comprises asequence of nucleotides that encode a therapeutic product that inhibitsan immune checkpoint or other immune suppressing target. Targetsinclude, but are not limited to, TREX1, PD-L1, VISTA, TGF-beta isoform1, beta-catenin, SIRP-alpha, VEGF, RNase H2, DNase II,CLEVER-1/Stabilin-1, and CD47. Other targets to be inhibited, suppressedor disrupted, are selected from among any of CTLA-4, PD-L2, PD-1, PD-2,IDO1, IDO2, LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, Galectin-9, CEACAM1,CD155, CD112, CD226, CD244 (2B4), B7-H2, B7-H3, ICOS, GITR, B7-H4,B7-H6, CD27, CD40, CD40L, CD48, CD70, CD80, CD86, CD137 (4-1BB), CD200,CD272 (BTLA), CD160, CD39, CD73, A2a receptor, A2b receptor, HHLA2,ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2/4, OX40, OX-40L, KIR, TIM1,TIM4, and STAT3. Exemplary thereof are among human PD-L1 (SEQ ID NO:31),human beta-catenin (SEQ ID NO:32), human SIRPa (SEQ ID NO:33), humanTREX1 (SEQ ID NO:34), human VISTA (SEQ ID NO:35), human TGF-beta isoform1 (SEQ ID NO: 193), and human VEGF (SEQ ID NO: 194). RNA can target orcontain a sequence in the immune checkpoint nucleic acids set forth inany of SEQ ID NOs: 1-30, 36-40, and 195-217. The plasmids in any of theimmunostimulatory bacteria also can encode a sequence of nucleotidesthat is an agonist of retinoic acid-inducible gene I (RIG-I) or a RIG-Ibinding element.

The immunostimulatory bacteria can include one or more of deletions ingenes, for example, the bacteria can be one or more of purI⁻ (purM⁻),msbB⁻, purD⁻, flagellin⁻ (fliC⁻/fljB⁻), pagP⁻, adrA⁻, csgD⁻ and hilA⁻.The immunostimulatory bacteria can be msbB⁻. For example, theimmunostimulatory bacteria can contain a purI deletion, an msbBdeletion, an asd deletion, an adrA deletion, and optionally, a csgDdeletion. Exemplary of bacterial gene deletions/modifications are any ofthe following:

one or more of a mutation in a gene that alters the biosynthesis oflipopolysaccharide, selected from among one or more of rfaL, rfaG, rfaH,rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA,and lpxT; and/or

one or more of a mutation that introduces a suicide gene and is selectedfrom one or more of sacB, nuk, hok, gef kil or phlA; and/or

one or more of a mutation that introduces a bacterial lysis gene and isselected from one or both of hly and cly; and/or

a mutation in one or more virulence factor(s), selected from among IsyA,pag, prg, iscA, virG, plc and act; and/or

one or more of a mutation in a gene that modifies the stress response,selected from among recA, htrA, htpR, hsp and groEL; and/or

a mutation in min that disrupts the cell cycle; and/or

one or more mutations in genes that disrupt or inactivate regulatoryfunctions, selected from among cya, crp, phoP/phoQ, and ompR.

The immunostimulatory bacterium can be a strain of Salmonella, Shigella,E. coli, Bifidobacteriae, Rickettsia, Vibrio, Listeria, Klebsiella,Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium,Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium,Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter,Bacillus, or Erysipelothrix, or an attenuated strain thereof or modifiedstrain thereof of any of the preceding list of bacterial strains.

Exemplary of the immunostimulatory bacteria are those where the plasmidcontains one or more of a sequence of nucleic acids encoding alisteriolysin O (LLO) protein lacking the signal sequence (cytoLLO), aCpG motif, a DNA nuclear targeting sequence (DTS), and a retinoicacid-inducible gene-I (RIG-I) binding element.

Other exemplary immunostimulatory bacteria include those that areauxotrophic for adenosine, and comprise: a deletion in the gene(s)encoding the flagella; a deletion in endA; a plasmid that encodesCytoLLO; a nuclear localization sequence; and an asd plasmidcomplementation system; and encode RNA that inhibits, suppresses ordisrupts expression of an immune checkpoint or other target whoseinhibition, suppression or disruption increases the anti-tumor immuneresponse in a subject.

Such immunostimulatory bacteria include strains of Salmonella, such as awild type Salmonella typhimurium strain, such as the strain depositedunder ATCC accession no. 14028, or a strain having all of theidentifying characteristics of the strain deposited under ATCC accession#14028. Other strains include, for example, an attenuated Salmonellatyphimurium strain selected from among strains designated as AST-100,VNP20009, or strains YS1646 (ATCC #202165), RE88, SL7207, χ 8429, χ8431, and χ 8468.

The immunostimulatory bacteria can contain one or more of a purI⁻deletion, an msbB deletion, an asd deletion, and an adrA deletion, inaddition to the modifications that increase accumulation in tumor cells,the TME, and/or tumor-resident immune cells, and/or modifications thatreduce immune cell death, and can encode an immunostimulatory protein orother therapeutic product as described herein. The immunostimulatorybacteria also can include:

one or more of a mutation in a gene that alters the biosynthesis oflipopolysaccharide selected from among one or more of rfaL, rfaG, rfaH,rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA,and lpxT; and/or

one or more of a mutation that introduces a suicide gene and is selectedfrom among one or more of sacB, nuk, hok, gef kil and phlA; and/or

one or more of a mutation that introduces a bacterial lysis gene and isselected from among one or both of hly and cly; and/or

a mutation in one or more virulence factor(s) selected from among IsyA,pag, prg, iscA, virG, plc and act; and/or

one or more mutations in a gene or genes that modify the stress responseselected from among recA, htrA, htpR, hsp and groEL; and/or

a mutation in min that disrupts the cell cycle; and/or

one or more mutations that disrupt or inactivate regulatory functionsselected from among cya, crp, phoP/phoQ and ompR.

The strains can be one or more of msbB⁻, asd⁻, hilA⁻ and/or flagellin⁻(fliC⁻/fljB⁻), and/or pagP⁻. In particular, the strains are flagellin⁻(fliC⁻/fljB⁻), such as flagellin⁻ (fliC⁻/fljB⁻), msbB⁻, purI⁻/M⁻, andoptionally asd⁻ and/or HilA⁻. The bacteria can be auxotrophic foradenosine, or adenosine and adenine. The therapeutic product, such asRNAi and/or an immunostimulatory protein, and/or antibody or fragmentthereof, are expressed under control of a promoter recognized by thehost, such as an RNAP III promoter or an RNAP II promoter, as describedherein. The immunostimulatory bacterium can be a strain of Salmonella,Shigella, E. coli, Bifidobacteriae, Rickettsia, Vibrio, Listeria,Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera,Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella,Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas,Helicobacter, Bacillus, or Erysipelothrix, or an attenuated strainthereof or modified strain thereof of any of the preceding list ofbacterial strains. Generally, the strain is one that is attenuated inthe host. Salmonella strains, such as S. typhimurium, are exemplary ofthe bacteria. Exemplary strains include Salmonella typhimurium strainsderived from strains designated as AST100, VNP20009, or strains YS1646(ATCC #202165), RE88, SL7207, χ 8429, χ 8431, χ 8468, and the wild-typestrain ATCC #14028.

Compositions containing the immunostimulatory bacteria are provided.Such compositions contain the bacteria and a pharmaceutically acceptableexcipient or vehicle. The immunostimulatory bacteria include anydescribed herein or in patents/applications incorporated herein or knownto those of skill in the art. The bacteria encode a therapeutic product,generally an anti-cancer product such as an inhibitor of an immunecheckpoint or an immunostimulatory protein that increases anti-tumoractivity in the tumor microenvironment or in the tumor, such as acytokine or chemokine or co-stimulatory molecule. The genomes of thebacteria can be modified to have increased infectivity of immune cells,and or reduced infectivity of non-immune cells, and/or reduced abilityto induce cell death of immune cells. Hence, the bacteria are modifiedas described herein to accumulate in tumors or the tumormicroenvironment or tumor-resident immune cells, and/or to deliverimmunostimulatory proteins and other therapeutic products that promoteanti-tumor activity. The immunostimulatory bacteria can additionallycontain a plasmid encoding a therapeutic anti-cancer product, such asRNAi, such as miRNA or shRNA, or a CRISPR cassette, that target animmune checkpoint, or otherwise enhance the anti-tumor activity of thebacteria.

A single dose is therapeutically effective for treating a disease ordisorder in which immune stimulation effects treatment. Exemplary ofsuch stimulation is an immune response, that includes, but is notlimited to, one or both of a specific immune response and non-specificimmune response, both specific and non-specific immune responses, aninnate response, a primary immune response, adaptive immunity, asecondary immune response, a memory immune response, immune cellactivation, immune cell proliferation, immune cell differentiation, andcytokine expression.

Pharmaceutical compositions containing any of the immunostimulatorybacteria are provided. As are uses thereof for treatment of cancers, andmethods of treatment of cancer. Methods and uses include treating asubject who has cancer, comprising administering an immunostimulatorybacterium or the pharmaceutical composition to a subject, such as ahuman. A method of treating a subject who has cancer, comprisingadministering an immunostimulatory bacterium, is provided.

Methods and uses include combination therapy in which a secondanti-cancer agent or treatment is administered. The second anti-canceragent is a chemotherapeutic agent that results in cytosolic DNA, orradiotherapy, or an anti-immune checkpoint inhibitor, such as ananti-PD-1, or anti-PD-L1 or anti-CTLA4 antibody, or CAR-T cells or othertherapeutic cells, such as stem cells, TIL cells and modified cells forcancer therapy. The combination therapy also can include anti-VEGF oranti-VEGFR, or anti-VEGFR2 antibodies, or fragments thereof, or ananti-IL6 antibody or fragment thereof, or oncolytic virus therapy, or acancer vaccine.

Administration can be by any suitable route, such as parenteral, and caninclude additional agents that can facilitate or enhance delivery.Administration can be oral or rectal or by aerosol into the lung, or canbe intratumorally, intravenously, intramuscularly, or subcutaneously.

Cancers include solid tumors and hematologic malignancies, such as, butnot limited to, lymphoma, leukemia, gastric cancer, and cancer of thebreast, heart, lung, small intestine, colon, spleen, kidney, bladder,head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone,bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

The immunostimulatory bacteria can be formulated into compositions foradministration, such as suspensions. They can be dried and stored aspowders. Combinations of the immunostimulatory bacteria with otheranti-cancer agents also are provided.

Combination therapies for treatment of cancers and malignancies areprovided. The immunostimulatory bacteria can be administered before, orconcurrently with, other cancer therapies, including radiotherapy,chemotherapies, particularly genotoxic chemotherapies that result incytosolic DNA, and immunotherapies, such as anti-checkpoint inhibitorantibodies, including anti-PD-1 antibodies, anti-PD-L1 antibodies, andanti-CTLA4 antibodies, and other such immunotherapies. Other cancertherapies also include anti-VEGF, anti-VEGFR, anti-VEGFR2, or anti-IL6antibodies, or fragments thereof, cancer vaccines and oncolytic viruses.

Administration can be by any suitable route, including systemic or localor topical, such as parenteral, including, for example, oral or rectalor by aerosol into the lung, or intratumorally, intravenously,intramuscularly, or subcutaneously.

Also provided are methods for increasing the colonization of tumors,tumor-resident immune cells, and/or the tumor microenvironment by animmunostimulatory bacterium. The methods include, for example, modifyingthe genome of a bacterium to render the bacterium flagellin⁻(fliC⁻/fljB⁻) and/or pagP⁻. It is shown herein that such modification(s)strikingly enhance tumor/tumor microenvironment/tumor-resident immunecell colonization.

The terms and expressions that are employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions to exclude any equivalents of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of the process used to delete the asd genefrom strain YS1646. The asd gene from S. typhimurium strain YS1646 wasdeleted using lambda-derived Red recombination system as described inDatsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)).

FIG. 2 depicts the levels of tumor colonization in injected and distaltumors after IT administration of AST-104. BALB/c mice (6-8 week old)were implanted with dual CT26 (2×10⁵ cells) subcutaneous flank tumors onthe right and left flanks (n=10 per group). Mice with established tumorswere IT injected into the right flank with 5×10⁶ CFU of the YS1646strain containing a TREX1 shRNA plasmid (AST-104). At 35 days post tumorimplantation (12 days after the last dose of AST-104), three mice weresacrificed, and injected and distal tumors were homogenized(GentleMACs™, Miltenyi Biotec) and plated on LB plates to enumerate thenumber of colony forming units (CFU) per gram of tumor tissue. Thefigure depicts the mean CFU per gram of tissue, +SD.

FIG. 3 depicts that CpG scrambled plasmid has immuno-stimulatoryanti-tumor properties. BALB/c mice (6-8 week old) were implanted with asingle CT26 (2×10⁵ cells) subcutaneous flank tumor (n=9 per group). Micewith established tumors were IV injected with 5×10⁶ CFU of the YS1646strain (AST-100), or the YS1646 strain containing the scrambled shRNAcontrol plasmid (AST-103), or PBS control, on the days indicated by thearrows. Tumor measurements were performed using electronic calipers(Fowler, Newton, Mass.). Tumor volume was calculated using the modifiedellipsoid formula ½(length×width²). Mice were euthanized when tumor sizereached >20% of body weight or became necrotic, as per IACUCregulations. TGI is calculated as 1-(mean test tumor volume/mean controltumor volume)×100. The figure depicts mean tumor growth of each group,±SEM. **p<0.01, student's t-test.

FIG. 4 depicts a schematic of the process used to delete the fliC gene.The flic gene was deleted from the chromosome of S. typhimurium strainAST-101 (asd deleted strain of YS1646) using lambda-derived Redrecombination system as described in Datsenko and Wanner (Proc. Natl.Acad. Sci. USA 97:6640-6645 (2000)).

FIG. 5 depicts that the flagellin deletion strain grows normally in LB.The figure depicts the growth of strains AST-108 ASD (pATI-shTREX1) andAST-112 ASD/FLG (pATI-shTREX1) at 37° C. in LB broth, as measured byOD₆₀₀ using a Spectramax 96 well plate reader (Molecular devices).

FIG. 6 depicts that flagellin knockout improves anti-tumor efficacy.BALB/c mice (6-8 week old) were implanted with a single CT26 (2×10⁵cells) subcutaneous flank tumor (n=9 per group). Mice with establishedtumors were IV injected with 5×10⁶ CFU of the asd/fljB/fliC knockoutstrain containing the pATI shTREX1 plasmid (AST-113), or asd knockoutstrain containing the pATI shTREX1 plasmid (AST-110), or PBS control, onthe days indicated by the arrows. Tumor measurements were performedusing electronic calipers (Fowler, Newton, Mass.). Tumor volume wascalculated using the modified ellipsoid formula ½(length×width²). Micewere euthanized when tumor size reached >20% of body weight or becamenecrotic, as per IACUC regulations. TGI was calculated as 1-(mean testtumor volume/mean control tumor volume)×100. The figure depicts the meantumor growth of each group, ±SEM. *p<0.05, student's t-test.

FIG. 7 depicts that flagellin knockout shows an increased IFN-gammasignature. BALB/c mice (6-8 week old) were implanted with a single CT26(2×10⁵ cells) subcutaneous flank tumor (n=9 per group). Mice withestablished tumors were IV injected with 5×10⁶ CFU of the asd/fljB/fliCknockout strain containing the pATI shTREX1 plasmid (AST-113), or asdknockout strain containing the pATI shTREX1 plasmid (AST-110), or PBScontrol. Mice were bled 6 hours following the first dose and systemicserum cytokines tested by Luminex 200 device (Luminex Corporation) andmouse cytometric bead array (BD bead array, FACS Fortessa, FCAPsoftware, all BD Biosciences). *p<0.05, **p<0.01, ***p<0.001, student'st-test.

FIG. 8 depicts that flagellin is not required for tumor colonization.BALB/c mice (6-8 week old) were implanted with a single CT26 (2×10⁵cells) subcutaneous flank tumor (n=9 per group). Mice with establishedtumors were IV injected with 5×10⁶ CFU of the asd/fljB/fliC knockoutstrain containing the pATI shTREX1 plasmid (AST-113), or asd knockoutstrain containing the pATI shTREX1 plasmid (AST-110), or PBS control. At35 days (D35) post tumor implantation (12 days after the last dose ofengineered Salmonella therapy), three mice per group were sacrificed,and tumors were homogenized (GentleMACs™, Miltenyi Biotec) and plated onLB plates to enumerate the number of colony forming units per gram oftumor tissue. The figure depicts the mean colony forming units (CFU) pergram of tissue, ±SD.

FIG. 9 depicts that a cytoLLO expressing strain grows normally in vitro.The figure depicts the growth of strains AST-110 (YS1646 with asddeletion containing (pATI-shTREX1)) and AST-115 (YS1646 with asddeletion and knock-in of cytoLLO expression cassette containing(pATI-shTREX1)) at 37° C. in LB broth, as measured by OD₆₀₀ using aSpectramax 96 well plate reader (Molecular devices).

FIG. 10 depicts that AST-115 (ASD knockout+CytoLLO Knock-in straincarrying shTREX1 plasmid) demonstrates potent, single-dose efficacy in amurine CT26 tumor model. BALB/c mice (6-8 week old) were implanted witha single CT26 (2×10⁵ cells) subcutaneous flank tumor (n=9 per group).Mice with established tumors were IV injected with 5×10⁶ CFU of AST-115(YS1646 with asd deletion and knock-in of cytoLLO expression cassette atasd locus containing (pATI-shTREX1)), or PBS control, on the daysindicated by the arrows. Tumor measurements were performed usingelectronic calipers (Fowler, Newton, Mass.). Tumor volume was calculatedusing the modified ellipsoid formula ½(length×width²). Mice wereeuthanized when tumor size reached >20% of body weight or becamenecrotic, as per IACUC regulations. TGI was calculated as 1-(mean testtumor volume/mean control tumor volume)×100. The figure depicts the meantumor growth of each group, ±SEM. **p <0.01, student's t-test.

FIG. 11 depicts that strain YS1646 requires tumor microenvironmentlevels of adenosine for growth. Growth of strains YS1646 (purI⁻/msbB⁻)and the wild-type parental strain ATCC 14028 at 37° C. in LB broth areshown, as measured by OD₆₀₀ using a Spectramax 96 well plate reader(Molecular devices).

FIG. 12 depicts that ASD, FLG, and CytoLLO engineered strains requirehigh adenosine for growth. The growth of strains AST-117 (YS1646 Δasdcontaining a low copy shTREX-1 plasmid), AST-118 (YS1646Δasd/ΔfliC/ΔfljB containing a low copy shTREX-1 plasmid), and AST-119(YS1646 Δasd:LLO containing a low copy shTREX-1 plasmid) at 37° C. in LBbroth are shown, as measured by OD₆₀₀ using a Spectramax 96 well platereader (Molecular devices).

FIG. 13 depicts that a strain with a low copy origin of replicationasd-encoding plasmid has superior growth kinetics than a strain with ahigh copy origin of replication asd-encoding plasmid. The growth ofstrains YS1646, AST-117 (YS1646 Δasd containing a low copy shTREX-1plasmid with a functional asd gene), AST-104 (YS1646 containing a lowcopy pEQ shTREX-1 plasmid without an asd gene), and AST-110 (YS1646 Δasdcontaining a high copy pATI-shTREX-1 plasmid with a functional asd gene)at 37° C. in LB broth are shown, as measured by OD₆₀₀ using a Spectramax96 well plate reader (Molecular devices).

FIG. 14 depicts that a strain with a low copy asd plasmid is more fitthan a strain with a high copy asd plasmid in mouse tumor cells. Theintracellular growth of strains AST-117 (YS1646 Δasd containing a lowcopy shTREX-1 plasmid with a functional asd gene) and AST-110 (YS1646Δasd containing a high copy pATI-shTREX-1 plasmid with a functional asdgene) are shown in B16F. 10 mouse melanoma cells and CT26 mouse coloncarcinoma cells. 5×10⁵ cells in a 24 well dish were infected with the S.typhimurium strains at a MOI of 5. After 30 minutes of infection, mediawas replaced with media containing gentamycin to kill extracellularbacteria. At indicated time points, cell monolayers were lysed byosmotic shock the cell lysates were diluted and plated on LB agar toenumerate CFU.

FIG. 15 depicts that in vivo, asd gene complementation systems result inretention of plasmids in S. typhimurium-infected tumors. BALB/c mice(6-8 week old) were implanted with a single CT26 (2×10⁵ cells)subcutaneous flank tumor (n=9 per group). Mice with established tumorswere IV injected with 5×10⁶ CFU of the asd knockout strain containingthe pATI shTREX1 plasmid (AST-110) or the YS1646 containing a pEQshTREX-1 plasmid without an asd gene (AST-104). At 35 days post tumorimplantation (12 days after the last dose of engineered Salmonellatherapy), three mice per group were sacrificed, and tumors werehomogenized using a GentleMACs™ homogenizer (Miltenyi Biotec) and platedon LB agar plates or LB agar plates with 50 ug/mL of Kanamycin. Thefigure depicts the percentage of Kanamycin resistant CFU in tumor tissuehomogenates, ±SD.

FIG. 16 depicts that the therapeutic efficacy of a strain containing aplasmid with asd gene complementation system and shTREX1 (AST-110) isimproved. BALB/c mice (6-8 week old) were implanted with a single CT26(2×10⁵ cells) subcutaneous flank tumor (n=9 per group). Mice withestablished tumors were IV injected with 5×10⁶ CFU of the asd knockoutstrain containing the pATI-shTREX1 plasmid (AST-110) or the asd knockoutstrain containing the pATI-scramble plasmid (AST-109), or the YS1646strain containing a pEQ-shTREX-1 plasmid without an asd gene (AST-104),or PBS control, on the days indicated by the arrows. Tumor measurementswere performed using electronic calipers (Fowler, Newton, Mass.). Tumorvolume was calculated using the modified ellipsoid formula½(length×width²). Mice were euthanized when tumor size reaches >20% ofbody weight or became necrotic, as per IACUC regulations. TGI wascalculated as 1-(mean test tumor volume/mean control tumor volume)×100.The figure depicts the mean tumor growth of each group, ±SEM.

FIG. 17 depicts that a strain containing a low copy shTREX1 plasmid(AST-117) has superior anti-tumor properties compared to a straincontaining a high copy plasmid (AST-110). BALB/c mice (6-8 week old)were implanted with a single CT26 (2×10⁵ cells) subcutaneous flank tumor(n=9 per group). Mice with established tumors were IV injected with5×10⁶ CFU of the asd knockout strain containing the pATI-shTREX1 plasmidwith a high copy number origin of replication (AST-110) or the asdknockout strain containing the pATI-shTREX1 plasmid with a low copynumber origin of replication (AST-117), or PBS control, on the daysindicated by the arrows. Tumor measurements were performed usingelectronic calipers (Fowler, Newton, Mass.). Tumor volume was calculatedusing the modified ellipsoid formula ½(length×width²). Mice wereeuthanized when tumor size reached >20% of body weight or becamenecrotic, as per IACUC regulations. TGI was calculated as 1-(mean testtumor volume/mean control tumor volume)×100. The figure depicts the meantumor growth of each group, ±SEM. *p<0.05, student's t-test.

FIGS. 18A and 18B depict that the AST-117 low copy plasmid straincolonizes tumors better and has a higher tumor to spleen colonizationratio than the AST-110 high copy plasmid strain. BALB/c mice (6-8 weekold) were implanted with a single CT26 (2×10⁵ cells) subcutaneous flanktumor (n=9 per group). Mice with established tumors were IV injectedwith 5×10⁶ CFU of the asd knockout strain containing the pATI-shTREX1plasmid with a high copy number origin of replication (AST-110) or theasd knockout strain containing the pATI-shTREX1 plasmid with a low copynumber origin of replication (AST-117). At 35 days post tumorimplantation (12 days after the last dose of engineered Salmonellatherapy), 3 mice per group were sacrificed, and tumors were homogenizedusing a GentleMACs™ homogenizer (Miltenyi Biotec) and plated on LBplates to enumerate the number of CFU per gram of tumor tissue. FIG. 18Adepicts the mean CFU per gram of tumor tissue, +SD. FIG. 18B depicts thetumor to spleen colonization ratios.

FIG. 19 depicts that autolytic strain (AST-120) cannot grow in theabsence of DAP. The figure depicts the growth of Δasd:cytoLLO straincontaining a pEQU6-shTREX1 plasmid that does not contain an asd gene(AST-120) over time in LB broth alone, or in LB broth supplemented with50 μg/mL DAP, as measured by OD₆₀₀ using a Spectramax 96 well platereader (Molecular devices).

FIG. 20 depicts the anti-tumor activity of the autolytic strain(AST-120). BALB/c mice (6-8 week old) were implanted with a single CT26(2×10⁵ cells) subcutaneous flank tumor (n=9 per group). Mice withestablished tumors were IV injected with 5×10⁶ CFU of the of Δasd:cytoLLO strain containing a pEQU6-shTREX1 plasmid that does not containan asd gene (AST-120), or PBS control, on the days indicated by thearrows. Tumor measurements were performed using electronic calipers(Fowler, Newton, Mass.). Tumor volume was calculated using the modifiedellipsoid formula ½(length×width²). Mice were euthanized when tumor sizereached >20% of body weight or became necrotic, as per IACUCregulations. TGI was calculated as 1-(mean test tumor volume/meancontrol tumor volume)×100. The figure depicts the mean tumor growth ofeach group, ±SEM. *p<0.05, student's t-test.

FIG. 21 depicts proteins that act downstream of HilA in the SPI-1pathway.

FIG. 22 depicts the SPI-1 T3SS and the functional classification ofSPI-1 encoded proteins (adapted from Kimbrough and Miller (2002)Microbes Infect. 4(1):75-82).

FIG. 23 depicts the effects of the SPI-1 T3SS on macrophages. Flagellinis detected by NAIP5/6 and the rod and needle proteins are detected byNAIP1/2, which leads to activation of the NLRC4 inflammasome andcaspase-1, resulting in the release of IL-1β and IL-18, and pyroptosis.

FIG. 24 depicts T3SS-1-mediated entry of the bacterium into theepithelial cell and the SCV.

FIG. 25 depicts recognition of bacterial flagellin by TLR5 and LPS byTLR4, and the role that flagellin, LPS, HilA, PrgI and PrgJ play in hostcell infection, cytokine release, inflammasome activation, andpyroptosis.

DETAILED DESCRIPTION

OUTLINE

-   -   A. DEFINITIONS    -   B. OVERVIEW OF THE IMMUNOSTIMULATORY BACTERIA    -   C. CANCER IMMUNOTHERAPEUTICS        -   1. Immunotherapies        -   2. Adoptive Immunotherapies        -   3. Cancer Vaccines and Oncolytic Viruses    -   D. BACTERIAL CANCER IMMUNOTHERAPY        -   1. Bacterial Therapies        -   2. Comparison of the Immune Responses to Bacteria and            Viruses        -   3. Salmonella Therapy            -   a. Tumor-tropic Bacteria.            -   b. Salmonella enterica Serovar Typhimurium            -   c. Bacterial Attenuation                -   i. msbB Mutants                -   ii. purI⁻ Mutants                -   iii. Combinations of Attenuating Mutations                -   iv. VNP20009 and Other Attenuated and Wild-type S.                    typhimurium Strains                -   v. S. typhimurium Engineered To Deliver                    Macromolecules        -   4. Enhancements of Immunostimulatory Bacteria to Increase            Therapeutic Index            -   a. asd Gene Deletion            -   b. Adenosine Auxotrophy            -   c. Flagellin Deficient Strains            -   d. Salmonella Engineered to Escape the Salmonella                Containing Vacuole (SCV)            -   e. Deletions in Salmonella Genes Required for Biofilm                Formation            -   f. Deletions in Genes in the LPS Biosynthetic Pathway            -   g. Deletions of SPI-1 and SPI-2 Genes            -   h. Endonuclease (endA) Mutations To Increase Plasmid                Delivery            -   i. RIG-I Inhibition            -   j. DNase II Inhibition            -   k. RNase H2 Inhibition            -   l. Stabilin-1/CLEVER-1 Inhibition        -   5. Immunostimulatory Proteins        -   6. Modifications that Increase Uptake of Gram-negative            Bacteria, such as Salmonella, by Immune Cells and Reduce            Immune Cell Death        -   7. Bacterial Culture Conditions    -   E. BACTERIAL ATTENUATION AND COLONIZATION        -   1. Deletion of flagellin (fliC/fljB)        -   2. Deletion of Genes in the LPS Biosynthetic Pathway        -   3. Colonization    -   F. CONSTRUCTING EXEMPLARY PLASMIDS ENCODING THERAPEUTIC PROTEINS        -   1. Immunostimulatory Proteins        -   2. Antibodies and Antibody Fragments        -   3. Interfering RNAs (RNAi)            -   a. shRNA            -   b. MicroRNA        -   4. Origin of Replication and Plasmid Copy Number        -   5. CpG Motifs and CpG Islands        -   6. Plasmid Maintenance/Selection Components        -   7. RNA Polymerase Promoters        -   8. DNA Nuclear Targeting Sequences        -   9. CRISPR    -   G. TUMOR-TARGETING IMMUNOSTIMULATORY BACTERIA CONTAIN RNAI        AGAINST EXEMPLARY IMMUNE TARGET GENES TO STIMULATE ANTI-TUMOR        IMMUNITY        -   1. TREX1        -   2. PD-L1        -   3. VISTA        -   4. SIRPα        -   5. ρ-catenin        -   6. TGF-β        -   7. VEGF        -   8. Additional Exemplary Checkpoint Targets    -   H. PHARMACEUTICAL PRODUCTION, COMPOSITIONS, AND FORMULATIONS        -   1. Manufacturing            -   a. Cell Bank Manufacturing            -   b. Drug Substance Manufacturing            -   c. Drug Product Manufacturing        -   2. Compositions        -   3. Formulations            -   a. Liquids, Injectables, Emulsions            -   b. Dried Thermostable Formulations        -   4. Compositions for Other Routes of Administration        -   5. Dosages and Administration        -   6. Packaging and Articles of Manufacture    -   I. METHODS OF TREATMENT AND USES        -   1. Tumors        -   2. Administration        -   3. Monitoring    -   J. EXAMPLES

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GenBank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, therapeutic bacteria are bacteria that effect therapy,such as cancer or anti-tumor therapy, when administered to a subject,such as a human.

As used herein, immunostimulatory bacteria are therapeutic bacteriathat, when introduced into a subject, accumulate in immunoprivilegedtissues and cells, such as tumors, and replicate and/or express productsthat are immunostimulatory or that result in immunostimulation. Forexample, the immunostimulatory bacteria are attenuated in the host byvirtue of reduced toxicity or pathogenicity and/or by virtue of encodedproducts that reduce toxicity or pathogenicity, as the immunostimulatorybacteria cannot replicate and/or express products (or have reducedreplication/product expression), except primarily in immunoprivilegedenvironments. Immunostimulatory bacteria provided herein are modified toencode a product or products or exhibit a trait or property that rendersthem immunostimulatory. Such products, properties and traits include,but are not limited to, for example, at least one of: animmunostimulatory protein, such as a cytokine, chemokine orco-stimulatory molecule; RNAi, such as siRNA (shRNA and microRNA), orCRISPR, that targets, disrupts or inhibits an immune checkpoint genesuch as TREX1 and/or PD-L1; or an inhibitor of an immune checkpoint suchas an anti-immune checkpoint antibody. Immunostimulatory bacteria alsocan include a modification that renders the bacterium auxotrophic for ametabolite that is immunosuppressive or that is in an immunosuppressivepathway, such as adenosine.

As used herein, the strain designations VNP20009 (see, e.g.,International PCT Application Publication No. WO 99/13053, see, alsoU.S. Pat. No. 6,863,894) and YS1646 and 41.2.9 are used interchangeablyand each refer to the strain deposited with the American Type CultureCollection (ATCC) and assigned Accession No. 202165. VNP20009 is amodified attenuated strain of Salmonella typhimurium, which containsdeletions in msbB and purI, and was generated from wild type strain ATCC#14028.

As used herein, the strain designations YS1456 and 8.7 are usedinterchangeably and each refer to the strain deposited with the AmericanType Culture Collection and assigned Accession No. 202164 (see, U.S.Pat. No. 6,863,894).

As used herein, an origin of replication is a sequence of DNA at whichreplication is initiated on a chromosome, plasmid or virus. For smallDNA, including bacterial plasmids and small viruses, a single origin issufficient.

The origin of replication determines the vector copy number, whichdepends upon the selected origin of replication. For example, if theexpression vector is derived from the low-copy-number plasmid pBR322, itis between about 25-50 copies/cell, and if derived from thehigh-copy-number plasmid pUC, it can be 150-200 copies/cell.

As used herein, medium copy number of a plasmid in cells is about or is150 or less than 150, low copy number is 15-30, such as 20 or less than20. Low to medium copy number is less than 150. High copy number isgreater than 150 copies/cell.

As used herein, a CpG motif is a pattern of bases that include anunmethylated central CpG (“p” refers to the phosphodiester link betweenconsecutive C and G nucleotides) surrounded by at least one baseflanking (on the 3′ and the 5′ side of) the central CpG. A CpGoligodeoxynucleotide is an oligodeoxynucleotide that is at least aboutten nucleotides in length and includes an unmethylated CpG. At least theC of the 5′ CG 3′ is unmethylated.

As used herein, a RIG-I binding sequence refers to a 5′triphosphate(5′ppp) structure directly, or that which is synthesized by RNA pol IIIfrom a poly(dA-dT) sequence, which by virtue of interaction with RIG-Ican activate type I IFN via the RIG-I pathway. The RNA includes at leastfour A ribonucleotides (A-A-A-A); it can contain 4, 5, 6, 7, 8, 9, 10 ormore. The RIG-I binding sequence is introduced into a plasmid in thebacterium for transcription into the polyA.

As used herein, an immunostimulatory protein is one that confers,promotes, enhance or increases immune responses, particularly in thetumor microenvironment, such as in tumors and/or in tumor-residentimmune cells. Immunostimulatory proteins include, but are not limitedto, cytokines, chemokines, co-stimulatory molecules, and other immuneregulatory proteins and products. Thus, as used herein, an“immunostimulatory protein” is a protein that confers, exhibits orpromotes an anti-tumor immune response in the tumor microenvironment.Exemplary of such proteins are cytokines, chemokines, and co-stimulatorymolecules, such as, but not limited to, GM-CSF, IL-2, IL-7, IL-12,IL-15, IL-18, IL-21, IL-12p70 (IL-12p40+IL-12p35), IL-15/IL-15R alphachain complex, CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, moleculesinvolved in the potential recruitment/persistence of T cells, CD40, CD40ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL),members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptideantagonists, and members of the TNFR superfamily.

As used herein, “cytokines” are a broad and loose category of smallproteins (˜5-20 kDa) that are important in cell signaling. Cytokinesinclude chemokines, interferons, interleukins, lymphokines, and tumornecrosis factors. Cytokines are cell signaling molecules that aid cellto cell communication in immune responses, and stimulate the movement ofcells towards sites of inflammation, infection and trauma.

As used herein, “chemokines” refer to chemoattractant (chemotactic)cytokines that bind to chemokine receptors and include proteins isolatedfrom natural sources as well as those made synthetically, as byrecombinant means or by chemical synthesis. Exemplary chemokinesinclude, but are not limited to, IL-8, IL-10, GCP-2, GRO-α, GRO-β,GRO-γ, ENA-78, PBP, CTAP III, NAP-2, LAPF-4, MIG (CXCL9), CXCL10,CXCL11, PF4, IP-10, SDF-1α, SDF-1β, SDF-2, MCP-1, MCP-2, MCP-3, MCP-4,MCP-5, MIP-1α (CCL3), MIP-1β (CCL4), MIP-1γ, MIP-2, MIP-2α, MIP-3α,MIP-3β, MIP-4, MIP-5, MDC, HCC-1, ALP, lungkine, Tim-1, eotaxin-1,eotaxin-2, I-309, SCYA17, TRAC, RANTES (CCL5), DC-CK-1, lymphotactin,ALP, lungkine and fractalkine, and others known to those of skill in theart. Chemokines are involved in the migration of immune cells to sitesof inflammation, as well as in the maturation of immune cells and in thegeneration of adaptive immune responses.

As used herein, a bacterium that is modified so that it “induces lesscell death in tumor-resident immune cells” or “induces less cell deathin immune cells” is one that is less toxic than the bacterium withoutthe modification, or one that has reduced virulence compared to thebacterium without the modification. Exemplary of such modifications arethose that decrease/eliminate pyroptosis and that alterlipopolysaccharide (LPS) profiles on the bacterium. These modificationsinclude one or more of disruption of or deletion of flagellin genes,pagP, or one or more components of the SPI-1 pathway, such as hilA, rodprotein, needle protein, and QseC.

As used herein, a bacterium that is “modified so that it preferentiallyinfects tumor-resident immune cells” or “modified so that itpreferentially infects immune cells” has a modification in its genomethat reduces its ability to infect cells other than immune cells.Exemplary of such modifications are modifications that disrupt the type3 secretion system or type 4 secretion system or other genes or systemsthat affect the ability of a bacterium to invade a non-immune cell. Forexample, disruption/deletion of an SPI-1 component, which is needed forinfection of cells, such as epithelial cells, but does not affectinfection of immune cells, such as phagocytic cells, by Salmonella.

As used herein, a “modification” is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids or nucleotides, respectively. Methods ofmodifying a polypeptide are routine to those of skill in the art, suchas by using recombinant DNA methodologies.

As used herein, a modification to a bacterial genome or to a plasmid orgene includes deletions, replacements and insertions of nucleic acid.

As used herein, RNA interference (RNAi) is a biological process in whichRNA molecules inhibit gene expression or translation, by neutralizingtargeted mRNA molecules to inhibit translation and thereby expression ofa targeted gene.

As used herein, RNA molecules that act via RNAi are referred to asinhibitory by virtue of their silencing of expression of a targetedgene. Silencing expression means that expression of the targeted gene isreduced or suppressed or inhibited.

As used herein, gene silencing via RNAi is said to inhibit, suppress,disrupt or silence expression of a targeted gene. A targeted genecontains sequences of nucleotides that correspond to the sequences inthe inhibitory RNA, whereby the inhibitory RNA silences expression ofmRNA.

As used herein, inhibiting, suppressing, disrupting or silencing atargeted gene refers to processes that alter expression, such astranslation, of the targeted gene, whereby activity or expression of theproduct encoded by the targeted gene is reduced. Reduction, includes acomplete knock-out or a partial knockout, whereby with reference to theimmunostimulatory bacterium provided herein and administration herein,treatment is effected.

As used herein, small interfering RNAs (siRNAs) are small pieces ofdouble-stranded (ds) RNA, usually about 21 nucleotides long, with 3′overhangs (2 nucleotides) at each end that can be used to “interfere”with the translation of proteins by binding to and promoting thedegradation of messenger RNA (mRNA) at specific sequences. In doing so,siRNAs prevent the production of specific proteins based on thenucleotide sequences of their corresponding mRNAs. The process is calledRNA interference (RNAi), and also is referred to as siRNA silencing orsiRNA knockdown.

As used herein, a short-hairpin RNA or small-hairpin RNA (shRNA) is anartificial RNA molecule with a tight hairpin turn that can be used tosilence target gene expression via RNA interference (RNAi). Expressionof shRNA in cells is typically accomplished by delivery of plasmids orthrough viral or bacterial vectors.

As used herein, a tumor microenvironment (TME) is the cellularenvironment in which the tumor exists, including surrounding bloodvessels, immune cells, fibroblasts, bone marrow-derived inflammatorycells, lymphocytes, signaling molecules and the extracellular matrix(ECM). Conditions that exist include, but are not limited to, increasedvascularization, hypoxia, low pH, increased lactate concentration,increased pyruvate concentration, increased interstitial fluid pressureand altered metabolites or metabolism, such as higher levels ofadenosine, indicative of a tumor.

As used herein, human type I interferons (IFNs) are a subgroup ofinterferon proteins that regulate the activity of the immune system. Alltype I IFNs bind to a specific cell surface receptor complex, such asthe IFN-α receptor. Type I interferons include IFN-α and IFN-β, amongothers. IFN-β proteins are produced by fibroblasts, and have antiviralactivity that is involved mainly in innate immune response. Two types ofIFN-β are IFN-β1 (IFNB1) and IFN-β3 (IFNB3).

As used herein, recitation that a nucleic acid or encoded RNA targets agene means that it inhibits or suppresses or silences expression of thegene by any mechanism. Generally, such nucleic acid includes at least aportion complementary to the targeted gene, where the portion issufficient to form a hybrid with the complementary portion.

As used herein, “deletion,” when referring to a nucleic acid orpolypeptide sequence, refers to the deletion of one or more nucleotidesor amino acids compared to a sequence, such as a target polynucleotideor polypeptide or a native or wild-type sequence.

As used herein, “insertion,” when referring to a nucleic acid or aminoacid sequence, describes the inclusion of one or more additionalnucleotides or amino acids, within a target, native, wild-type or otherrelated sequence. Thus, a nucleic acid molecule that contains one ormore insertions compared to a wild-type sequence, contains one or moreadditional nucleotides within the linear length of the sequence.

As used herein, “additions” to nucleic acid and amino acid sequencesdescribe addition of nucleotides or amino acids onto either terminicompared to another sequence.

As used herein, “substitution” or “replacement” refers to the replacingof one or more nucleotides or amino acids in a native, target, wild-typeor other nucleic acid or polypeptide sequence with an alternativenucleotide or amino acid, without changing the length (as described innumbers of residues) of the molecule. Thus, one or more substitutions ina molecule does not change the number of amino acid residues ornucleotides of the molecule. Amino acid replacements compared to aparticular polypeptide can be expressed in terms of the number of theamino acid residue along the length of the polypeptide sequence.

As used herein, “at a position corresponding to,” or recitation thatnucleotides or amino acid positions “correspond to” nucleotides or aminoacid positions in a disclosed sequence, such as set forth in theSequence Listing, refers to nucleotides or amino acid positionsidentified upon alignment with the disclosed sequence to maximizeidentity using a standard alignment algorithm, such as the GAPalgorithm. By aligning the sequences, one skilled in the art canidentify corresponding residues, for example, using conserved andidentical amino acid residues as guides. In general, to identifycorresponding positions, the sequences of amino acids are aligned sothat the highest order match is obtained (see, e.g., ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,eds., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAMJ Applied Math 48:1073).

As used herein, alignment of a sequence refers to the use of homology toalign two or more sequences of nucleotides or amino acids. Typically,two or more sequences that are related by 50% or more identity arealigned. An aligned set of sequences refers to 2 or more sequences thatare aligned at corresponding positions and can include aligningsequences derived from RNAs, such as ESTs and other cDNAs, aligned withgenomic DNA sequence. Related or variant polypeptides or nucleic acidmolecules can be aligned by any method known to those of skill in theart. Such methods typically maximize matches, and include methods, suchas using manual alignments and by using the numerous alignment programsavailable (e.g., BLASTP) and others known to those of skill in the art.By aligning the sequences of polypeptides or nucleic acids, one skilledin the art can identify analogous portions or positions, using conservedand identical amino acid residues as guides. Further, one skilled in theart also can employ conserved amino acid or nucleotide residues asguides to find corresponding amino acid or nucleotide residues betweenand among human and non-human sequences. Corresponding positions alsocan be based on structural alignments, for example by using computersimulated alignments of protein structure. In other instances,corresponding regions can be identified. One skilled in the art also canemploy conserved amino acid residues as guides to find correspondingamino acid residues between and among human and non-human sequences.

As used herein, a “property” of a polypeptide, such as an antibody,refers to any property exhibited by a polypeptide, including, but notlimited to, binding specificity, structural configuration orconformation, protein stability, resistance to proteolysis,conformational stability, thermal tolerance, and tolerance to pHconditions. Changes in properties can alter an “activity” of thepolypeptide. For example, a change in the binding specificity of theantibody polypeptide can alter the ability to bind an antigen, and/orvarious binding activities, such as affinity or avidity, or in vivoactivities of the polypeptide.

As used herein, an “activity” or a “functional activity” of apolypeptide, such as an antibody, refers to any activity exhibited bythe polypeptide. Such activities can be empirically determined.Exemplary activities include, but are not limited to, ability tointeract with a biomolecule, for example, through antigen-binding, DNAbinding, ligand binding, or dimerization, or enzymatic activity, forexample, kinase activity or proteolytic activity. For an antibody(including antibody fragments), activities include, but are not limitedto, the ability to specifically bind a particular antigen, affinity ofantigen-binding (e.g., high or low affinity), avidity of antigen-binding(e.g., high or low avidity), on-rate, off-rate, effector functions, suchas the ability to promote antigen neutralization or clearance, virusneutralization, and in vivo activities, such as the ability to preventinfection or invasion of a pathogen, or to promote clearance, or topenetrate a particular tissue or fluid or cell in the body. Activity canbe assessed in vitro or in vivo using recognized assays, such as ELISA,flow cytometry, surface plasmon resonance or equivalent assays tomeasure on- or off-rate, immunohistochemistry and immunofluorescencehistology and microscopy, cell-based assays, flow cytometry and bindingassays (e.g., panning assays).

As used herein, “bind,” “bound” or grammatical variations thereof refersto the participation of a molecule in any attractive interaction withanother molecule, resulting in a stable association in which the twomolecules are in close proximity to one another. Binding includes, butis not limited to, non-covalent bonds, covalent bonds (such asreversible and irreversible covalent bonds), and includes interactionsbetween molecules such as, but not limited to, proteins, nucleic acids,carbohydrates, lipids, and small molecules, such as chemical compoundsincluding drugs.

As used herein, “antibody” refers to immunoglobulins and immunoglobulinfragments, whether natural or partially or wholly synthetic, such asrecombinantly produced, including any fragment thereof containing atleast a portion of the variable heavy chain and light region of theimmunoglobulin molecule that is sufficient to form an antigen-bindingsite and, when assembled, to specifically bind an antigen. Hence, anantibody includes any protein having a binding domain that is homologousor substantially homologous to an immunoglobulin antigen-binding domain(antibody combining site). For example, an antibody refers to anantibody that contains two heavy chains (which can be denoted H and H′)and two light chains (which can be denoted L and L′), where each heavychain can be a full-length immunoglobulin heavy chain or a portionthereof sufficient to form an antigen binding site (e.g., heavy chainsinclude, but are not limited to, VH chains, VH-CH1 chains andVH-CH1-CH2-CH3 chains), and each light chain can be a full-length lightchain or a portion thereof sufficient to form an antigen binding site(e.g., light chains include, but are not limited to, VL chains and VL-CLchains). Each heavy chain (H and H′) pairs with one light chain (L andL′, respectively). Typically, antibodies minimally include all or atleast a portion of the variable heavy (VH) chain and/or the variablelight (VL) chain. The antibody also can include all or a portion of theconstant region.

For purposes herein, the term antibody includes full-length antibodiesand portions thereof, including antibody fragments, such as anti-EGFRantibody fragments. Antibody fragments, include, but are not limited to,Fab fragments, Fab′ fragments, F(ab′)₂ fragments, a nanobody (such as acamelid antibody), fragments, disulfide-linked Fvs (dsFv), Fd fragments,Fd′ fragments, single-chain Fvs (scFv), single-chain Fabs (scFab),diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-bindingfragments of any of the above. Antibody also includes syntheticantibodies, recombinantly produced antibodies, multispecific antibodies(e.g., bispecific antibodies), human antibodies, non-human antibodies,humanized antibodies, chimeric antibodies, and intrabodies. Antibodiesprovided herein include members of any immunoglobulin class (e.g., IgG,IgM, IgD, IgE, IgA and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or sub-subclass (e.g., IgG2a and IgG2b). Antibodies forhuman therapy generally are human antibodies or are humanized.

As used herein, “antibody fragment(s)” refers to (i) monovalent andmonospecific antibody derivatives that contain the variable heavy and/orlight chains or functional fragments of an antibody and lack an Fc part;and (ii) BiTE (tandem scFv), DARTs, diabodies and single-chain diabodies(scDB). Thus, an antibody fragment includes a/an: Fab, Fab′, scFab,scFv, Fv fragment, nanobody (see, e.g., antibodies derived from Camelusbactriamus, Calelus dromaderius, or Lama paccos) (see, e.g., U.S. Pat.No. 5,759,808; and Stijlemans et al. (2004) J. Biol. Chem.279:1256-1261), VHH, dAb, minimal recognition unit, single-chain diabody(scDb), BiTE and DART. The recited antibody fragments have a molecularweight below 60 kDa.

As used herein, “nucleic acid” refers to at least two linked nucleotidesor nucleotide derivatives, including a deoxyribonucleic acid (DNA) and aribonucleic acid (RNA), joined together, typically by phosphodiesterlinkages. Also included in the term “nucleic acid” are analogs ofnucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA,and other such analogs and derivatives or combinations thereof. Nucleicacids also include DNA and RNA derivatives containing, for example, anucleotide analog or a “backbone” bond other than a phosphodiester bond,for example, a phosphotriester bond, a phosphoramidate bond, aphosphorothioate bond, a thioester bond, or a peptide bond (peptidenucleic acid). The term also includes, as equivalents, derivatives,variants and analogs of either RNA or DNA made from nucleotide analogs,single (sense or antisense) and double-stranded nucleic acids.Deoxyribonucleotides include deoxyadenosine, deoxycytidine,deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.

As used herein, an isolated nucleic acid molecule is one which isseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid molecule. An “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Exemplary isolated nucleic acidmolecules provided herein include isolated nucleic acid moleculesencoding an antibody or antigen-binding fragments provided.

As used herein, “operably linked” with reference to nucleic acidsequences, regions, elements or domains means that the nucleic acidregions are functionally related to each other. For example, a nucleicacid encoding a leader peptide can be operably linked to a nucleic acidencoding a polypeptide, whereby the nucleic acids can be transcribed andtranslated to express a functional fusion protein, wherein the leaderpeptide effects secretion of the fusion polypeptide. In some instances,the nucleic acid encoding a first polypeptide (e.g., a leader peptide)is operably linked to a nucleic acid encoding a second polypeptide andthe nucleic acids are transcribed as a single mRNA transcript, buttranslation of the mRNA transcript can result in one of two polypeptidesbeing expressed. For example, an amber stop codon can be located betweenthe nucleic acid encoding the first polypeptide and the nucleic acidencoding the second polypeptide, such that, when introduced into apartial amber suppressor cell, the resulting single mRNA transcript canbe translated to produce either a fusion protein containing the firstand second polypeptides, or can be translated to produce only the firstpolypeptide. In another example, a promoter can be operably linked tonucleic acid encoding a polypeptide, whereby the promoter regulates ormediates the transcription of the nucleic acid.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, “polypeptide” refers to two or more amino acidscovalently joined. The terms “polypeptide” and “protein” are usedinterchangeably herein.

As used herein, a “peptide” refers to a polypeptide that is from 2 toabout or 40 amino acids in length.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids contained in theantibodies provided include the twenty naturally-occurring amino acids(see Table below), non-natural amino acids, and amino acid analogs(e.g., amino acids wherein the α-carbon has a side chain). As usedherein, the amino acids, which occur in the various amino acid sequencesof polypeptides appearing herein, are identified according to theirwell-known, three-letter or one-letter abbreviations (see Table below).The nucleotides, which occur in the various nucleic acid molecules andfragments, are designated with the standard single-letter designationsused routinely in the art.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are generally in the“L” isomeric form. Residues in the “D” isomeric form can be substitutedfor any L-amino acid residue, as long as the desired functional propertyis retained by the polypeptide. NH₂ refers to the free amino grouppresent at the amino terminus of a polypeptide. COOH refers to the freecarboxy group present at the carboxyl terminus of a polypeptide. Inkeeping with standard polypeptide nomenclature described in J. Biol.Chem., 243:3557-59 (1968) and adopted at 37 C.F.R. §§ 1.821-1.822,abbreviations for amino acid residues are shown in the following Table:

Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID Y TyrTyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glutamic Acid and/or Glutamine W Trp TryptophanR Arg Arginine D Asp Aspartic acid N Asn Asparagine B Asx Aspartic Acidand/or Asparagine C Cys Cysteine X Xaa Unknown or other

All sequences of amino acid residues represented herein by a formulahave a left to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. The phrase “amino acid residue” isdefined to include the amino acids listed in the above Table ofCorrespondence, modified, non-natural and unusual amino acids. A dash atthe beginning or end of an amino acid residue sequence indicates apeptide bond to a further sequence of one or more amino acid residues orto an amino-terminal group such as NH₂ or to a carboxyl-terminal groupsuch as COOH.

In a peptide or protein, suitable conservative substitutions of aminoacids are known to those of skill in the art and generally can be madewithout altering a biological activity of a resulting molecule. Those ofskill in the art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.,Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. Co., p. 224).

Such substitutions can be made in accordance with the exemplarysubstitutions set forth in the following Table:

Exemplary Conservative Amino Acid Substitutions

Exemplary Original Conservative residue substitution(s) Ala (A) Gly; SerArg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G)Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T)Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu

Other substitutions also are permissible and can be determinedempirically or in accord with other known conservative ornon-conservative substitutions.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, the term “non-natural amino acid” refers to an organiccompound that has a structure similar to a natural amino acid but hasbeen modified structurally to mimic the structure and reactivity of anatural amino acid. Non-naturally occurring amino acids thus include,for example, amino acids or analogs of amino acids other than the 20naturally occurring amino acids and include, but are not limited to, theD-stereoisomers of amino acids. Exemplary non-natural amino acids areknown to those of skill in the art, and include, but are not limited to,2-Aminoadipic acid (Aad), 3-Aminoadipic acid (bAad),β-alanine/β-Amino-propionic acid (Bala), 2-Aminobutyric acid (Abu),4-Aminobutyric acid/piperidinic acid (4Abu), 6-Aminocaproic acid (Acp),2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib),3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm),2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2′-Diaminopimelic acid(Dpm), 2,3-Diaminopropionic acid (Dpr), N-Ethylglycine (EtGly),N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl), allo-Hydroxylysine(Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline (4Hyp), Isodesmosine(Ide), allo-Isoleucine (Aile), N-Methylglycine, sarcosine (MeGly),N-Methylisoleucine (MeIle), 6-N-Methyllysine (MeLys), N-Methylvaline(MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine (Orn).

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule cannot be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, production by recombinant methods refers means the useof the well-known methods of molecular biology for expressing proteinsencoded by cloned DNA.

As used herein, “heterologous nucleic acid” is nucleic acid that encodesproducts (i.e., RNA and/or proteins) that are not normally produced invivo by the cell in which it is expressed, or nucleic acid that is in alocus in which it does not normally occur, or that mediates or encodesmediators that alter expression of endogenous nucleic acid, such as DNA,by affecting transcription, translation, or other regulatablebiochemical processes. Heterologous nucleic acid, such as DNA, also isreferred to as foreign nucleic acid. Any nucleic acid, such as DNA, thatone of skill in the art would recognize or consider as heterologous orforeign to the cell in which it is expressed, is herein encompassed byheterologous nucleic acid; heterologous nucleic acid includesexogenously added nucleic acid that is also expressed endogenously.Heterologous nucleic acid is generally not endogenous to the cell intowhich it is introduced, but has been obtained from another cell orprepared synthetically or is introduced into a genomic locus in which itdoes not occur naturally, or its expression is under the control ofregulatory sequences or a sequence that differs from the naturalregulatory sequence or sequences.

Examples of heterologous nucleic acid herein include, but are notlimited to, nucleic acid that encodes RNAi, or an immunostimulatoryprotein, such as a cytokine, that confers or contributes to anti-tumorimmunity in the tumor microenvironment, or that encodes an antibody,antibody fragment or other therapeutic product or therapeutic protein.In the immunostimulatory bacteria, the heterologous nucleic acidgenerally is encoded on the introduced plasmid, but it can be introducedinto the genome of the bacterium, such as a promoter that altersexpression of a bacterial product. Heterologous nucleic acid, such asDNA, includes nucleic acid that can, in some manner, mediate expressionof DNA that encodes a therapeutic product, or it can encode a product,such as a peptide or RNA, that in some manner mediates, directly orindirectly, expression of a therapeutic product.

As used herein, cell therapy involves the delivery of cells to a subjectto treat a disease or condition. The cells, which can be allogeneic orautologous, are modified ex vivo, such as by infection of cells withimmunostimulatory bacteria provided herein, so that they deliver orexpress products when introduced to a subject.

As used herein, genetic therapy involves the transfer of heterologousnucleic acid, such as DNA, into certain cells, such as target cells, ofa mammal, particularly a human, with a disorder or condition for whichsuch therapy is sought. The nucleic acid, such as DNA, is introducedinto the selected target cells in a manner such that the heterologousnucleic acid, such as DNA, is expressed and a therapeutic product(s)encoded thereby is produced. Genetic therapy can also be used to delivernucleic acid encoding a gene product that replaces a defective gene orsupplements a gene product produced by the mammal or the cell in whichit is introduced. The introduced nucleic acid can encode a therapeuticcompound, such as a growth factor or inhibitor thereof, or a tumornecrosis factor or inhibitor thereof, such as a receptor thereof, thatis not normally produced in the mammalian host or that is not producedin therapeutically effective amounts or at a therapeutically usefultime. The heterologous nucleic acid, such as DNA, encoding thetherapeutic product, can be modified prior to introduction into thecells of the afflicted host in order to enhance or otherwise alter theproduct or expression thereof. Genetic therapy can also involve deliveryof an inhibitor or repressor or other modulator of gene expression.

As used herein, “expression” refers to the process by which polypeptidesare produced by transcription and translation of polynucleotides. Thelevel of expression of a polypeptide can be assessed using any methodknown in art, including, for example, methods of determining the amountof the polypeptide produced from the host cell. Such methods caninclude, but are not limited to, quantitation of the polypeptide in thecell lysate by ELISA, Coomassie blue staining following gelelectrophoresis, Lowry protein assay and Bradford protein assay.

As used herein, a “host cell” is a cell that is used to receive,maintain, reproduce and/or amplify a vector. A host cell also can beused to express the polypeptide encoded by the vector. The nucleic acidcontained in the vector is replicated when the host cell divides,thereby amplifying the nucleic acids.

As used herein, a “vector” is a replicable nucleic acid from which oneor more heterologous proteins, can be expressed when the vector istransformed into an appropriate host cell. Reference to a vectorincludes those vectors into which a nucleic acid encoding a polypeptideor fragment thereof can be introduced, typically by restriction digestand ligation. Reference to a vector also includes those vectors thatcontain nucleic acid encoding a polypeptide, such as a modifiedanti-EGFR antibody. The vector is used to introduce the nucleic acidencoding the polypeptide into the host cell for amplification of thenucleic acid or for expression/display of the polypeptide encoded by thenucleic acid. The vectors typically remain episomal, but can be designedto effect integration of a gene or portion thereof into a chromosome ofthe genome. Also contemplated are vectors that are artificialchromosomes, such as yeast artificial chromosomes and mammalianartificial chromosomes. Selection and use of such vehicles arewell-known to those of skill in the art. A vector also includes “virusvectors” or “viral vectors.” Viral vectors are engineered viruses thatare operatively linked to exogenous genes to transfer (as vehicles orshuttles) the exogenous genes into cells.

As used herein, an “expression vector” includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well-known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, “primary sequence” refers to the sequence of amino acidresidues in a polypeptide or the sequence of nucleotides in a nucleicacid molecule.

As used herein, “sequence identity” refers to the number of identical orsimilar amino acids or nucleotide bases in a comparison between a testand a reference polypeptide or polynucleotide. Sequence identity can bedetermined by sequence alignment of nucleic acid or protein sequences toidentify regions of similarity or identity. For purposes herein,sequence identity is generally determined by alignment to identifyidentical residues. The alignment can be local or global. Matches,mismatches and gaps can be identified between compared sequences. Gapsare null amino acids or nucleotides inserted between the residues ofaligned sequences so that identical or similar characters are aligned.Generally, there can be internal and terminal gaps. When using gappenalties, sequence identity can be determined with no penalty for endgaps (e.g., terminal gaps are not penalized). Alternatively, sequenceidentity can be determined without taking into account gaps as thenumber of identical positions/length of the total aligned sequence×100.

As used herein, a “global alignment” is an alignment that aligns twosequences from beginning to end, aligning each letter in each sequenceonly once. An alignment is produced, regardless of whether or not thereis similarity or identity between the sequences. For example, 50%sequence identity based on “global alignment” means that in an alignmentof the full sequence of two compared sequences each of 100 nucleotidesin length, 50% of the residues are the same. It is understood thatglobal alignment also can be used in determining sequence identity evenwhen the length of the aligned sequences is not the same. Thedifferences in the terminal ends of the sequences will be taken intoaccount in determining sequence identity, unless the “no penalty for endgaps” is selected. Generally, a global alignment is used on sequencesthat share significant similarity over most of their length. Exemplaryalgorithms for performing global alignment include the Needleman-Wunschalgorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplaryprograms for performing global alignment are publicly available andinclude the Global Sequence Alignment Tool available at the NationalCenter for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/),and the program available atdeepc2.psi.iastate.edu/aat/align/align.html.

As used herein, a “local alignment” is an alignment that aligns twosequences, but only aligns those portions of the sequences that sharesimilarity or identity. Hence, a local alignment determines ifsub-segments of one sequence are present in another sequence. If thereis no similarity, no alignment will be returned. Local alignmentalgorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math.2: 482 (1981)). For example, 50% sequence identity based on “localalignment” means that in an alignment of the full sequence of twocompared sequences of any length, a region of similarity or identity of100 nucleotides in length has 50% of the residues that are the same inthe region of similarity or identity.

For purposes herein, sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Default parameters for the GAP program can include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) and the weighted comparison matrix of Gribskovet al. (1986) Nucl. Acids Res. 14: 6745, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Whether any two nucleic acid moleculeshave nucleotide sequences or any two polypeptides have amino acidsequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical,” or other similar variations reciting a percent identity,can be determined using known computer algorithms based on local orglobal alignment (see e.g.,wikipedia.org/wiki/Sequence_alignment_software, providing links todozens of known and publicly available alignment databases andprograms). Generally, for purposes herein sequence identity isdetermined using computer algorithms based on global alignment, such asthe Needleman-Wunsch Global Sequence Alignment tool available fromNCBI/BLAST(blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign(William Pearson implementing the Huang and Miller algorithm (Adv. Appl.Math. (1991) 12:337-357)); and program from Xiaoqui Huang available atdeepc2.psi.iastate.edu/aat/align/align.html. Typically, the full-lengthsequence of each of the compared polypeptides or nucleotides is alignedacross the full-length of each sequence in a global alignment. Localalignment also can be used when the sequences being compared aresubstantially the same length.

Therefore, as used herein, the term “identity” represents a comparisonor alignment between a test and a reference polypeptide orpolynucleotide. In one non-limiting example, “at least 90% identical to”refers to percent identities from 90 to 100% relative to the referencepolypeptide or polynucleotide. Identity at a level of 90% or more isindicative of the fact that, assuming for exemplification purposes atest and reference polypeptide or polynucleotide length of 100 aminoacids or nucleotides are compared, no more than 10% (i.e., 10 out of100) of amino acids or nucleotides in the test polypeptide orpolynucleotide differ from those of the reference polypeptide. Similarcomparisons can be made between a test and reference polynucleotides.Such differences can be represented as point mutations randomlydistributed over the entire length of an amino acid sequence or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g., 10/100 amino acid difference (approximately 90%identity). Differences also can be due to deletions or truncations ofamino acid residues. Differences are defined as nucleic acid or aminoacid substitutions, insertions or deletions. Depending on the length ofthe compared sequences, at the level of homologies or identities aboveabout 85-90%, the result can be independent of the program and gapparameters set; such high levels of identity can be assessed readily,often without relying on software.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment.

As used herein, treatment refers to any effects that ameliorate symptomsof a disease or disorder. Treatment encompasses prophylaxis, therapyand/or cure.

Treatment also encompasses any pharmaceutical use of anyimmunostimulatory bacterium or composition provided herein.

As used herein, prophylaxis refers to prevention of a potential diseaseand/or a prevention of worsening of symptoms or progression of adisease.

As used herein, “prevention” or prophylaxis, and grammaticallyequivalent forms thereof, refers to methods in which the risk orprobability of developing a disease or condition is reduced.

As used herein, a “pharmaceutically effective agent” includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, anesthetics, vasoconstrictors, dispersing agents, andconventional therapeutic drugs, including small molecule drugs andtherapeutic proteins.

As used herein, a “therapeutic effect” means an effect resulting fromtreatment of a subject that alters, typically improves or ameliorates,the symptoms of a disease or condition or that cures a disease orcondition.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect followingadministration to a subject. Hence, it is the quantity necessary forpreventing, curing, ameliorating, arresting or partially arresting asymptom of a disease or disorder.

As used herein, “therapeutic efficacy” refers to the ability of anagent, compound, material, or composition containing a compound toproduce a therapeutic effect in a subject to whom the agent, compound,material, or composition containing a compound has been administered.

As used herein, a “prophylactically effective amount” or a“prophylactically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset, or reoccurrence, of disease orsymptoms, reducing the likelihood of the onset, or reoccurrence, ofdisease or symptoms, or reducing the incidence of viral infection. Thefull prophylactic effect does not necessarily occur by administration ofone dose, and can occur only after administration of a series of doses.Thus, a prophylactically effective amount can be administered in one ormore administrations.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, an “anti-cancer agent” refers to any agent that isdestructive or toxic to malignant cells and tissues. For example,anti-cancer agents include agents that kill cancer cells or otherwiseinhibit or impair the growth of tumors or cancer cells. Exemplaryanti-cancer agents are chemotherapeutic agents.

As used herein “therapeutic activity” refers to the in vivo activity ofa therapeutic polypeptide. Generally, the therapeutic activity is theactivity that is associated with treatment of a disease or condition.

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a patient refers to a human subject.

As used herein, animal includes any animal, such as, but not limited to,primates including humans, gorillas and monkeys; rodents, such as miceand rats; fowl, such as chickens; ruminants, such as goats, cows, deer,sheep; pigs and other animals. Non-human animals exclude humans as thecontemplated animal. The polypeptides provided herein are from anysource, animal, plant, prokaryotic and fungal. Most polypeptides are ofanimal origin, including mammalian origin.

As used herein, a “composition” refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a “combination” refers to any association between oramong two or more items. The combination can be two or more separateitems, such as two compositions or two collections, a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related.

As used herein, combination therapy refers to administration of two ormore different therapeutics. The different therapeutics or therapeuticagents can be provided and administered separately, sequentially,intermittently, or can be provided in a single composition.

As used herein, a kit is a packaged combination that optionally includesother elements, such as additional reagents and instructions for use ofthe combination or elements thereof, for a purpose including, but notlimited to, activation, administration, diagnosis, and assessment of abiological activity or property.

As used herein, a “unit dose form” refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, a “single dosage formulation” refers to a formulationfor direct administration.

As used herein, a multi-dose formulation refers to a formulation thatcontains multiple doses of a therapeutic agent and that can be directlyadministered to provide several single doses of the therapeutic agent.The doses can be administered over the course of minutes, hours, weeks,days or months. Multi-dose formulations can allow dose adjustment,dose-pooling and/or dose-splitting. Because multi-dose formulations areused over time, they generally contain one or more preservatives toprevent microbial growth.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass any of the compositions provided herein contained in articlesof packaging.

As used herein, a “fluid” refers to any composition that can flow.Fluids thus encompass compositions that are in the form of semi-solids,pastes, solutions, aqueous mixtures, gels, lotions, creams and othersuch compositions.

As used herein, an isolated or purified polypeptide or protein (e.g., anisolated antibody or antigen-binding fragment thereof) orbiologically-active portion thereof (e.g., an isolated antigen-bindingfragment) is substantially free of cellular material or othercontaminating proteins from the cell or tissue from which the protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. Preparations can be determined tobe substantially free if they appear free of readily detectableimpurities as determined by standard methods of analysis, such as thinlayer chromatography (TLC), gel electrophoresis and high performanceliquid chromatography (HPLC), used by those of skill in the art toassess such purity, or sufficiently pure such that further purificationdoes not detectably alter the physical and chemical properties, such asenzymatic and biological activities, of the substance. Methods forpurification of the compounds to produce substantially chemically purecompounds are known to those of skill in the art. A substantiallychemically pure compound, however, can be a mixture of stereoisomers. Insuch instances, further purification might increase the specificactivity of the compound. As used herein, a “cellular extract” or“lysate” refers to a preparation or fraction which is made from a lysedor disrupted cell.

As used herein, a “control” refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a polypeptide, comprising “an immunoglobulindomain” includes polypeptides with one or a plurality of immunoglobulindomains.

As used herein, the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 amino acids” means “about 5 amino acids” and also “5 aminoacids.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally variantportion means that the portion is variant or non-variant.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, Biochem. (1972)11(9):1726-1732).

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. Overview of the Immunostimulatory Bacteria

Provided are modified bacteria, called immunostimulatory bacteria hereinthat accumulate and/or replicate in (i.e., colonize) tumors,tumor-resident immune cells and/or the tumor microenvironment (TME);and/or induce less cell death in tumor-resident immune cells; and/orencode therapeutic products, such as anti-tumor agents,immunostimulatory proteins, and inhibitory RNAs (RNAi), such as shRNAsand microRNAs (miRNAs), that target genes whose inhibition, suppressionor silencing effects tumor therapy. Strains of bacteria for modificationare any suitable for therapeutic use. The modified immunostimulatorybacteria provided herein are for uses and for methods for treatingcancer. The bacteria are modified for such uses and methods.

The immunostimulatory bacteria provided herein are modified by deletionor modification of bacterial genes to attenuate their inflammatoryresponses, increase their tolerability, increase their resistance tocomplement, increase their infectivity of, accumulation in orcolonization of tumors, tumor-resident immune cells and/or the TME,decrease their induction of immune cell death (e.g., decreasepyroptosis), and to enhance the anti-tumor immune responses in hoststreated with the bacteria. The modifications also can be in genesencoded on a plasmid in the bacteria. For example, the bacteria can beauxotrophic for adenosine, or adenosine and adenine, and plasmidsencoding therapeutic products, such as immunostimulatory proteins,antibodies, or RNAi that inhibit immune checkpoint genes in the host areincluded in the bacteria. Attenuation of the inflammatory responses tothe bacteria can be effected by deletion of the msbB gene, whichdecreases TNF-alpha in the host, and/or knocking out flagellin genesand/or deletion/mutation of pagP. The bacteria are modified to stimulatehost anti-tumor activity, for example, by adding plasmids encodingimmunostimulatory proteins such as cytokines, chemokines andco-stimulatory molecules, or RNAi that target host immune checkpoints,and by adding nucleic acid with CpGs/CpG motifs.

Bacterial strains can be attenuated strains, or strains that areattenuated by standard methods, or that, by virtue of the modificationsprovided herein, are attenuated in that their ability to colonize islimited primarily to immunoprivileged tissues and organs, particularlyimmune and tumor cells, including solid tumors. Bacteria include, butare not limited to, for example, strains of Salmonella, Shigella,Listeria, E. coli, and Bifidobacteriae. For example, species includeShigella sonnei, Shigella flexneri, Shigella disenteriae, Listeriamonocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonellagallinarum, and Salmonella enteritidis. Other suitable bacterial speciesinclude Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas,Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus,Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus,Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. Forexample, Rickettsia Rikettsiae, Rickettsia prowazekii, Rickettsiatsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetellabronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonaseucrenophila, Aeromonas salmonicida, Francisella tularensis,Corynebacterium pseudotuberculosis, Citrobacter feundii, Chlamydiapneumoniae, Haemophilus sornnus, Brucella abortus, Mycobacteriumintracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonasaeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis,Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaeaquintana, and Agrobacterium tumerfacium.

The bacteria accumulate by virtue of one or more properties, including,diffusion, migration and chemotaxis to immunoprivileged tissues ororgans or environments, environments that provide nutrients or othermolecules for which they are auxotrophic, and/or environments thatcontain replicating cells that provide environments suitable for entryand replication of the bacteria. The immunostimulatory bacteria providedherein and species that effect such therapy include species ofSalmonella, Listeria, and E. coli.

The bacteria contain plasmids that encode a therapeutic protein, such asan immunostimulatory protein or an immune checkpoint inhibitor or otherimmune stimulating or immune-suppression blocking product. Productsinclude antibodies, such as antibody fragments, and nanobodies, RNAi,such as one or more short hairpin (sh) RNA construct(s), microRNAs, orother RNAi modalities, whose expression inhibits or disrupts orsuppresses the expression of targeted genes, or otherwise increasesimmune responses or decreases immune suppression. The therapeuticproducts are expressed under control of a eukaryotic promoter, such asan RNA polymerase (RNAP) II or III promoter. Typically, RNAPIII (alsoreferred to as POLIII) promoters are constitutive, and RNAPII (alsoreferred to as POLII) can be regulated. In some examples, the shRNAstarget the gene TREX1, to inhibit its expression.

In some embodiments, the plasmids can encode a plurality of therapeuticproducts, including immunostimulatory proteins, such as cytokines, andRNAi molecules, such as shRNAs, and antibodies, including nanobodies,that inhibit two or more immune checkpoint genes, such as TREX1, PD-L1,VISTA, SIRPa, CTNNB1, TGF-beta, CD47, and/or VEGF and any others knownto those of skill in the art. Where a plurality of therapeutic productsare encoded, expression of each generally is under control of adifferent promoter.

Among the bacteria provided herein, are bacteria that are modified sothat they are auxotrophic for adenosine. This can be achieved bymodification or deletion of genes involved in purine synthesis,metabolism, or transport. For example, disruption of the tsx gene inSalmonella species, such as Salmonella typhi, results in adenosineauxotrophy. Adenosine is immunosuppressive and accumulates to highconcentrations in tumors; auxotrophy for adenosine improves theanti-tumor activity of the bacteria because the bacteria selectivelyreplicate in tissues rich in adenosine.

Also provided are bacteria that are modified so that they have adefective asd gene. These bacteria for use in vivo are modified toinclude carrying a functional asd gene on the introduced plasmid; thismaintains selection for the plasmid so that an antibiotic-based plasmidmaintenance/selection system is not needed. Also provided is the use ofasd defective strains that do not contain a functional asd gene on aplasmid, and are thus engineered to be autolytic in the host.

Also provided are bacteria that are modified so that they are incapableof producing flagella. This can be achieved by modifying the bacteria bydeleting the genes that encode the flagellin subunits. The modifiedbacteria lacking flagellin are less inflammatory and therefore bettertolerated, and induce a more potent anti-tumor response.

Also provided are bacteria that are modified to produce listeriolysin O(LLO), which improves plasmid delivery in phagocytic cells.

Also provided are bacteria modified to carry a low copy, CpG-containingplasmid. The plasmid further can include other modifications, and canencode therapeutic products, such as immunostimulatory proteins,antibodies and fragments thereof, and RNAi.

The bacteria also can be modified to grow in a manner such that thebacteria, if a Salmonella species, expresses less of the toxic SPI-1(Salmonella pathogenicity island-1) genes. In Salmonella, genesresponsible for virulence, invasion, survival, and extra intestinalspread are located in Salmonella pathogenicity islands (SPIs).

The bacteria include plasmids that encode RNAi, such as shRNA ormicroRNA, that inhibits checkpoints, such as PD-L1 or TREX1 only, orTREX1 and one or more of a second immune checkpoint. The bacteria can befurther modified for other desirable traits, including for selection ofplasmid maintenance, particularly for selection without antibiotics, forpreparation of the strains. The immunostimulatory bacteria optionallycan encode therapeutic polypeptides, including anti-tumor therapeuticpolypeptides and agents.

Exemplary of the immunostimulatory bacteria provided herein are speciesof Salmonella. Exemplary of bacteria for modification as describedherein are engineered strains of Salmonella typhimurium, such as strainYS1646 (ATCC Catalog #202165; also referred to as VNP20009, see,International PCT Application Publication No. WO 99/13053) that isengineered with plasmids to complement an asd gene knockout andantibiotic-free plasmid maintenance.

Modified immunostimulatory bacterial strains that are renderedauxotrophic for adenosine are provided herein, as are pharmaceuticalcompositions containing such strains, formulated for administration to asubject, such as a human, for use in methods of treating tumors andcancers.

Also provided are methods or uses of the immunostimulatory bacteria orpharmaceutical compositions containing the bacteria, wherein thetreatment comprises combination therapy, in which a second anti-canceragent or treatment is administered. The second anti-cancer agent ortreatment can be administered before, concomitantly with, after, orintermittently with, the immunostimulatory bacteria or pharmaceuticalcomposition, and includes immunotherapy, such as an antibody or antibodyfragment; oncolytic virus therapy; radiation/radiotherapy; andchemotherapy. The immunotherapy can comprise, for example,administration of an anti-PD-1, or anti-PD-L1 or anti-CTLA4, oranti-IL6, or anti-VEGF, or anti-VEGFR, or anti-VEGFR2 antibody, or afragment thereof. Combination therapy also can include surgery.

The engineered immunostimulatory bacteria provided herein containmultiple synergistic modalities to induce immune re-activation of coldtumors and to promote tumor antigen-specific immune responses, whileinhibiting immune checkpoint pathways that the tumor utilizes to subvertand evade durable anti-tumor immunity. Improved tumor targeting throughadenosine auxotrophy and enhanced vascular disruption have improvedpotency, while localizing the inflammation to limit systemic cytokineexposure and the autoimmune toxicities observed with other immunotherapymodalities. Exemplary of the bacteria so-modified are S. typhimuriumstrains, including such modifications of the strain YS1646, particularlyasd⁻ strains.

For example, as provided herein, are immunostimulatory bacteria thatprovide for shRNA-mediated gene disruption of PD-L1. It has been shownin mice that gene disruption of PD-L1 can improve tumor colonization. Ithas been shown, for example, that S. typhimurium infection in PD-L1knockout mice, results in a 10-fold higher bacterial load than inwild-type mice (see, Lee et al. (2010) Immunol. 185:2442-2449). Hence,PD-L1 is protective against S. typhimurium infection. Provided hereinare immunostimulatory bacteria, such as S. typhimurium, carryingplasmids capable of RNAi-mediated gene knockdown of TREX1, PD-L1, or ofPD-L1 and TREX1. Such bacteria provide anti-tumor effects due to thecombination of two independent pathways that lead to enhanced andsustained anti-tumor immune responses in a single therapy.

C. Cancer Immunotherapeutics

The immunosuppressive milieu found within the tumor microenvironment(TME) is a driver of tumor initiation and progression. Cancers emergeafter the immune system fails to control and contain tumors. Multipletumor-specific mechanisms create tumor environments wherein the immunesystem is forced to tolerate tumors and their cells instead ofeliminating them. The goal of cancer immunotherapy is to rescue theimmune system's natural ability to eliminate tumors. Acute inflammationassociated with microbial infection has been observationally linked withthe spontaneous elimination of tumors for centuries.

1. Immunotherapies

Several clinical cancer immunotherapies have sought to perturb thebalance of immune suppression towards anti-tumor immunity. Strategies tostimulate immunity through directly administering cytokines such as IL-2and IFN-α have seen modest clinical responses in a minority of patients,while inducing serious systemic inflammation-related toxicities (Sharmaet al. (2011) Nat. Rev. Cancer 11:805-812). The immune system hasevolved several checks and balances to limit autoimmunity, such asupregulation of programmed cell death protein 1 (PD-1) on T cells andits binding to its cognate ligand, programmed death-ligand 1 (PD-L1),which is expressed on both antigen presenting cells (APCs) and tumorcells. The binding of PD-L1 to PD-1 interferes with CD8⁺ T cellsignaling pathways, impairing the proliferation and effector function ofCD8⁺ T cells, and inducing T cell tolerance. PD-1 and PD-L1 are twoexamples of numerous inhibitory “immune checkpoints,” which function bydownregulating immune responses. Other inhibitory immune checkpointsinclude cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), signalregulatory protein a (SIRPa), V-domain Ig suppressor of T cellactivation (VISTA), programmed death-ligand 2 (PD-L2), indoleamine2,3-dioxygenase (IDO) 1 and 2, lymphocyte-activation gene 3 (LAG3),Galectin-9, T cell immunoreceptor with Ig and ITIM domains (TIGIT), Tcell immunoglobulin and mucin-domain containing-3 (TIM-3, also known ashepatitis A virus cellular receptor 2 (HAVCR2)), herpesvirus entrymediator (HVEM), CD39, CD73, B7-H3 (also known as CD276), B7-H4, CD47,CD48, CD80 (B7-1), CD86 (B7-2), CD155, CD160, CD244 (2B4), B- andT-lymphocyte attenuator (BTLA, or CD272) and carcinoembryonicantigen-related cell adhesion molecule 1 (CEACAM1, or CD66a).

Antibodies designed to block immune checkpoints, such as anti-PD-1 (forexample, pembrolizumab, nivolumab) and anti-PD-L1 (for example,atezolizumab, avelumab, durvalumab), have had durable success inpreventing T cell anergy and breaking immune tolerance. Only a fractionof treated patients demonstrate clinical benefit, and those that dooften present with autoimmune-related toxicities (see, e.g., Ribas(2015) N Engl J Med 373:1490-1492; Topalian et al. (2012) N Engl J Med366:3443-3447). This is further evidence for the need for therapies,provided herein, that are more effective and less toxic.

Another checkpoint blockade strategy inhibits the induction of CTLA-4 onT cells, which binds to and inhibits co-stimulatory receptors on APCs,such as CD80 or CD86, out-competing the co-stimulatory clusterdifferentiation 28 (CD28), which binds the same receptors, but with alower affinity. This blocks the stimulatory signal from CD28, while theinhibitory signal from CTLA-4 is transmitted, preventing T cellactivation (see, Phan et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100:8372-8377). Anti-CTLA-4 therapy (for example, ipilimumab) haveclinical success and durability in some patients, whilst exhibiting aneven greater incidence of severe immune-related adverse events (see,e.g., Hodi et al. (2010) N. Engl. J. Med. 363:711-723; Schadendorf etal. (2015) J. Clin. Oncol. 33:1889-1894). It also has been shown thattumors develop resistance to anti-immune checkpoint antibodies,highlighting the need for more durable anticancer therapies, andprovided herein. 2. Adoptive Immunotherapies In seeking to reactivate acold tumor to become more immunogenic, a class of immunotherapies knownas adoptive cell therapy (ACT) encompasses a variety of strategies toharness immune cells and reprogram them to have anti-tumor activity(Hinrichs et al. (2011) Immunol. Rev. 240:40-51). Dendritic cell-basedtherapies introduce genetically engineered dendritic cells (DCs) withmore immune-stimulatory properties. These therapies have not beensuccessful because they fail to break immune tolerance to cancer (see,e.g., Rosenberg et al. (2004) Nat. Med. 12:1279). A method using wholeirradiated tumor cells containing endogenous tumor antigens andgranulocyte macrophage colony-stimulating factor (GM-CSF) to stimulateDC recruitment, known as GVAX, similarly failed in the clinic due to thelack of ability to break tumor tolerance (Copier et al. (2010) Curr.Opin. Mol. Ther. 12:647-653). A separate autologous cell-based therapy,Sipuleucel-T (Provenge), was FDA approved in 2010 forcastration-resistant prostate cancer. It utilizes APCs retrieved fromthe patient and re-armed to express prostatic acid phosphatase (PAP)antigen to stimulate a T cell response, then re-introduced followinglymphablation. Unfortunately, its broader adoption has been limited bylow observed objective response rates and high costs, and its use islimited to only the early stages of prostate cancer (Anassi et al.(2011) P T. 36(4):197-202). Similarly, autologous T cell therapies(ATCs) harvest a patient's own T cells and reactivate them ex vivo toovercome tumor tolerance, then reintroduce them to the patient followinglymphablation. ATCs have had limited clinical success, and only inmelanoma, while generating serious safety and feasibility issues thatlimit their utility (Yee et al. (2013) Clin. Cancer Res. 19:1-3).

Chimeric antigen receptor T cell (CAR-T) therapies are T cells harvestedfrom patients that have been re-engineered to express a fusion proteinbetween the T cell receptor and an antibody Ig variable extracellulardomain. This confers upon them the antigen-recognition properties ofantibodies with the cytolytic properties of activated T cells (Sadelain(2015) Clin. Invest. 125:3392-4000). Success has been limited to B celland hematopoietic malignancies, at the cost of deadly immune-relatedadverse events (Jackson et al. (2016) Nat. Rev. Clin. Oncol.13:370-383). Tumors can also mutate to escape recognition by a targetantigen, including CD 19 (Ruella et al., (2016) Comput. Struct.Biotechnol. J. 14: 357-362) and EGFRvIII (O'Rourke et al. (2017) Sci.Transl. Med. July 19; 9:399), thereby fostering immune escape. Inaddition, while CAR-T therapies are approved and are approved in thecontext of hematological malignancies, they face a significant hurdlefor feasibility to treat solid tumors: overcoming the highlyimmunosuppressive nature of the solid tumor microenvironment. A numberof additional modifications to existing CAR-T therapies will be requiredto potentially provide feasibility against solid tumors (Kakarla et al.(2014) Cancer J. March-April; 20(2):151-155). When the safety of CAR-Tsis significantly improved and their efficacy expanded to solid tumors,the feasibility and costs associated with these labor-intensivetherapies will continue to limit their broader adoption.

3. Cancer Vaccines and Oncolytic Viruses

Cold tumors lack T cell and dendritic cell (DC) infiltration, and arenon-T-cell-inflamed (Sharma et al. (2017) Cell 9; 168(4):707-723). Inseeking to reactivate a cold tumor to become more immunogenic, anotherclass of immunotherapies harness microorganisms that can accumulate intumors, either naturally or by virtue of engineering. These includeviruses designed to stimulate the immune system to express tumorantigens, thereby activating and reprogramming the immune system toreject the tumor. Virally-based cancer vaccines have largely failedclinically for a number of factors, including pre-existing or acquiredimmunity to the viral vector itself, as well as a lack of sufficientimmunogenicity to the expressed tumor antigens (Larocca et al. (2011)Cancer J. 17(5):359-371). Lack of proper adjuvant activation of APCs hasalso hampered other non-viral vector cancer vaccines, such as DNAvaccines. Oncolytic viruses, in contrast, seek to preferentiallyreplicate in dividing tumor cells over healthy tissue, whereuponsubsequent tumor cell lysis leads to immunogenic tumor cell death andfurther viral dissemination. The oncolytic virus Talimogenelaherparepvec (T-VEC), which uses a modified herpes simplex virus incombination with the DC-recruiting cytokine GM-CSF, is FDA approved formetastatic melanoma (Bastin et al. (2016) Biomedicines 4(3):21). Whiledemonstrating clinical benefit in some melanoma patients, and with fewerimmune toxicities than with other immunotherapies, the intratumoralroute of administration and manufacturing conditions have been limiting,as well as its lack of distal tumor efficacy and broader application toother tumor types. Other oncolytic virus (OV)-based vaccines, such asthose utilizing paramyxovirus, reovirus and picornavirus, among others,have met with similar limitations in inducing systemic anti-tumorimmunity (Chiocca et al. (2014) Cancer Immunol. Res. 2(4):295-300).Systemic administration of oncolytic viruses presents unique challenges.Upon I.V. administration, the virus is rapidly diluted, thus requiringhigh titers that can lead to hepatotoxicity. Further, if pre-existingimmunity exists, the virus is rapidly neutralized in the blood, andacquired immunity then restricts repeat dosing (Maroun et al. (2017)Future Virol. 12(4):193-213).

Of the limitations of virally-based vaccine vectors and oncolyticviruses, the greatest limitations can be the virus itself. Viralantigens have strikingly higher affinities to human T cell receptors(TCR) compared to tumor antigens (Aleksic et al. (2012) Eur J. Immunol.42(12):3174-3179). Tumor antigens, presented alongside of viral vectorantigens by MHC-1 on the surface of even highly activated APCs, will beoutcompeted for binding to TCRs, resulting in very poor antigen-specificanti-tumor immunity. A tumor-targeting immunostimulatory vector, asprovided herein, that does not itself provide high affinity T cellepitopes can circumvent these limitations.

D. Bacterial Cancer Immunotherapy

1. Bacterial Therapies

The recognition that bacteria have anticancer activity goes back to the1800s, when several physicians observed regression of tumors in patientsinfected with Streptococcus pyogenes. William Coley began the firststudy utilizing bacteria for the treatment of end stage cancers, anddeveloped a vaccine composed of S. pyogenes and Serratia marcescens,which was successfully used to treat a variety of cancers, includingsarcomas, carcinomas, lymphomas and melanomas. Since then, a number ofbacteria, including species of Clostridium, Mycobacterium,Bifidobacterium, Listeria, such as, L. monocytogenes, and Escherichiaspecies, have been studied as sources of anti-cancer vaccines (see,e.g., International PCT Application Publication No. WO 1999/013053;International PCT Application Publication No. WO 2001/025399; Bermudeset al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199; Patyar et al.(2010) Journal of Biomedical Science 17:21; Pawelek et al. (2003) LancetOncol. 4:548-556).

Bacteria can infect animal and human cells, and some possess the innateability to deliver DNA into the cytosol of cells, and these arecandidate vectors for gene therapy. Bacteria also are suitable fortherapy because they can be administered orally, they propagate readilyin vitro and in vivo, and they can be stored and transported in alyophilized state. Bacterial genetics are readily manipulated, and thecomplete genomes for many strains have been fully characterized (Felgneret al. (2016) mbio 7(5):e01220-16). As a result, bacteria have been usedto deliver and express a wide variety of genes, including those thatencode cytokines, angiogenesis inhibitors, toxins and prodrug-convertingenzymes. Salmonella, for example, has been used to expressimmune-stimulating molecules like IL-18 (Loeffler et al. (2008) CancerGene Ther. 15(12):787-794), LIGHT (Loeffler et al. (2007) Proc. Natl.Acad. Sci. USA 104(31): 12879-12883), and Fas ligand (Loeffler et al.(2008) J. Natl. Cancer Inst. 100:1113-1116) in tumors. Bacterial vectorsalso are cheaper and easier to produce than viral vectors, and bacterialdelivery is favorable over viral delivery because it can be quicklyeliminated by antibiotics if necessary, rendering it a saferalternative.

To be used, however, the strains themselves must not be pathogenic orare not pathogenic after modification for use as a therapeutic. Forexample, in the treatment of cancer, the therapeutic bacterial strainsmust be attenuated or rendered sufficiently non-toxic so as to not causesystemic disease and/or septic shock, but still maintain some level ofinfectivity to effectively colonize tumors. Genetically modifiedbacteria have been described that are to be used as antitumor agents toelicit direct tumoricidal effects and/or to deliver tumoricidalmolecules (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002;Bermudes, D. et al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199;Zhao, M. et al. (2005) Proc. Natl. Acad. Sci. USA 102:755-760; Zhao, M.et al. (2006) Cancer Res. 66:7647-7652). Among these are bioengineeredstrains of Salmonella enterica Serovar typhimurium (S. typhimurium).These bacteria accumulate preferentially >1,000-fold greater in tumorsthan in normal tissues and disperse homogeneously in tumor tissues(Pawelek, J. et al. (1997) Cancer Res. 57:4537-4544; Low, K. B. et al.(1999) Nat. Biotechnol. 17:37-41). Preferential replication allows thebacteria to produce and deliver a variety of anticancer therapeuticagents at high concentrations directly within the tumor, whileminimizing toxicity to normal tissues. These attenuated bacteria aresafe in mice, pigs, and monkeys when administered intravenously (Zhao,M. et al. (2005) Proc. Natl. Acad. Sci. USA 102:755-760; Zhao, M. et al.(2006) Cancer Res 66:7647-7652; Tjuvajev J. et al. (2001) J. ControlRelease 74:313-315; Zheng, L. et al. (2000) Oncol. Res. 12:127-135), andcertain live attenuated Salmonella strains have been shown to be welltolerated after oral administration in human clinical trials (Chatfield,S. N. et al. (1992) Biotechnology 10:888-892; DiPetrillo, M. D. et al.(1999) Vaccine 18:449-459; Hohmann, E. L. et al. (1996) J. Infect. Dis.173:1408-1414; Sirard, J. C. et al. (1999) Immunol. Rev. 171:5-26). TheS. typhimurium phoP/phoQ operon is a typical bacterial two-componentregulatory system composed of a membrane-associated sensor kinase (PhoQ)and a cytoplasmic transcriptional regulator (PhoP: Miller, S. I. et al.(1989) Proc. Natl. Acad. Sci. USA 86:5054-5058; Groisman, E. A. et al.(1989) Proc. Natl. Acad. Sci. USA 86: 7077-7081). PhoP/phoQ is requiredfor virulence, and its deletion results in poor survival of thisbacterium in macrophages and a marked attenuation in mice and humans(Miller, S. I. et al. (1989) Proc. Natl. Acad. Sci. USA 86:5054-5058;Groisman, E. A. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 7077-7081;Galan, J. E. and Curtiss, R. III. (1989)Microb. Pathog. 6:433-443;Fields, P. I. et al. (1986) Proc. Natl. Acad. Sci. USA 83:189-193).PhoP/phoQ deletion strains have been employed as effective vaccinedelivery vehicles (Galan, J. E. and Curtiss, R. III. (1989) Microb.Pathog. 6:433-443; Fields, P. I. et al. (1986) Proc. Natl. Acad. Sci.USA 83:189-193; Angelakopoulos, H. and Hohmann, E. L. (2000) InfectImmun. 68:213-241). Attenuated Salmonellae have been used for targeteddelivery of tumoricidal proteins (Bermudes, D. et al. (2002) Curr. Opin.Drug Discov. Devel. 5:194-199; Tjuvajev J. et al. (2001) J ControlRelease 74:313-315).

Bacterially-based cancer therapies have demonstrated limited clinicalbenefit. A variety of bacterial species, including Clostridium novyi(Dang et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98(26):15155-15160;U.S. Patent Publications Nos. 2017/0020931, 2015/0147315; U.S. Pat. Nos.7,344,710; 3,936,354), Mycobacterium bovis (U.S. Patent Publication Nos.2015/0224151, 2015/0071873), Bifidobacterium bifidum (Kimura et al.(1980) Cancer Res. 40:2061-2068), Lactobacillus casei (Yasutake et al.(1984) Med. Microbiol. Immunol. 173(3):113-125), Listeria monocytogenes(Le et al. (2012) Clin. Cancer Res. 18(3):858-868; Starks et al. (2004)J. Immunol. 173:420-427; U.S. Patent Publication No. 2006/0051380) andEscherichia coli (U.S. Pat. No. 9,320,787) have been studied as possibleagents for anticancer therapy.

The Bacillus Calmette-Guerin (BCG) strain, for example, is approved forthe treatment of bladder cancer in humans, and is more effective thanintravesical chemotherapy, often being used as a first-line treatment(Gardlik et al. (2011) Gene therapy 18:425-431). Another approachutilizes Listeria monocytogenes, a live attenuated intracellularbacterium capable of inducing potent CD8⁺ T cell priming to expressedtumor antigens in mice (Le et al. (2012) Clin. Cancer Res.18(3):858-868). In a clinical trial of the Listeria-based vaccineincorporating the tumor antigen mesothelin, together with an allogeneicpancreatic cancer-based GVAX vaccine in a prime-boost approach, a mediansurvival of 6.1 months was noted in patients with advanced pancreaticcancer, versus a median survival of 3.9 months for patients treated withthe GVAX vaccine alone (Le et al. (2015) J. Clin. Oncol.33(12):1325-1333). These results were not replicated in a larger phase2b study, possibly pointing to the difficulties in attempting to induceimmunity to a low affinity self-antigen such as mesothelin.

Bacterial strains can be modified as described and exemplified herein toexpress inhibitory RNA (RNAi), such as shRNAs and microRNAs, thatinhibit or disrupt TREX1 and/or PD-L1 and optionally one or moreadditional immune checkpoint genes. The strains can be attenuated bystandard methods and/or by deletion or modification of genes, and byalteration or introduction of genes that render the bacteria able togrow in vivo primarily in immunoprivileged environments, such as theTME, in tumor cells and solid tumors. Strains for modification asdescribed herein can be selected from among, for example, Shigella,Listeria, E. coli, Bifidobacteriae and Salmonella. For example, Shigellasonnei, Shigella flexneri, Shigella disenteriae, Listeria monocytogenes,Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, andSalmonella enteritidis. Other suitable bacterial species includeRickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Franciesella,Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella,Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas,Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example,Rickettsia Rikettsiae, Rickettsia prowazecki, Rickettsia tsutsugamuchi,Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica,Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila,Aeromonas salmonicida, Franciesella tularensis, Corynebacteriumpseudotuberculosis, Citrobacter feundii, Chlamydia pneumoniae,Haemophilus sornnus, Brucella abortus, Mycobacterium intracellulare,Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa,Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis,Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaeaquintana, and Agrobacterium tumerfacium. Any known therapeutic,including immunostimulatory, bacteria can be modified as describedherein.

2. Comparison of the Immune Responses to Bacteria and Viruses

Bacteria, like viruses, have the advantage of being naturallyimmunostimulatory. Bacteria and viruses are known to contain conservedstructures known as Pathogen-Associated Molecular Patterns (PAMPs),which are sensed by host cell Pattern Recognition Receptors (PRRs).Recognition of PAMPs by PRRs triggers downstream signaling cascades thatresult in the induction of cytokines and chemokines, and initiation ofimmune responses that lead to pathogen clearance (Iwasaki and Medzhitov(2010) Science 327(5963):291-295). The manner in which the innate immunesystem is engaged by PAMPs, and from what type of infectious agent,determines the appropriate adaptive immune response to combat theinvading pathogen.

A class of PRRs known as Toll Like Receptors (TLRs) recognize PAMPsderived from bacterial and viral origins, and are located in variouscompartments within the cell. TLRs bind a range of ligands, includinglipopolysaccharide (TLR4), lipoproteins (TLR2), flagellin (TLR5),unmethylated CpG motifs in DNA (TLR9), double-stranded RNA (TLR3), andsingle-stranded RNA (TLR7 and TLR8) (Akira et al. (2001) Nat. Immunol.2(8):675-680; Kawai and Akira (2005) Curr. Opin. Immunol.17(4):338-344). Host surveillance of S. typhimurium for example, islargely mediated through TLR2, TLR4 and TLR5 (Arpaia et al. (2011) Cell144(5):675-688). These TLRs signal through MyD88 and TRIF adaptormolecules to mediate induction of NF-kB dependent pro-inflammatorycytokines such as TNF-α, IL-6 and IFN-γ (Pandey et al. (2015) ColdSpring Harb. Perspect. Biol. 7(1):a016246).

Another category of PRRs are the nod-like receptor (NLR) family. Thesereceptors reside in the cytosol of host cells and recognizeintracellular PAMPS. For example, S. typhimurium flagellin was shown toactivate the NLRC4/NAIP5 inflammasome pathway, resulting in the cleavageof caspase-1 and induction of the pro-inflammatory cytokines IL-1β andIL-18, leading to pyroptotic cell death of infected macrophages (Fink etal. (2007) Cell Microbiol. 9(11):2562-2570).

While engagement of TLR2, TLR4, TLR5 and the inflammasome inducespro-inflammatory cytokines that mediate bacterial clearance, theyactivate a predominantly NF-κB-driven signaling cascade that leads torecruitment and activation of neutrophils, macrophages and CD4⁺ T cells,but not the DCs and CD8⁺ T cells that are required for anti-tumorimmunity (Lui et al. (2017) Signal Transduct. Target Ther. 2:17023). Inorder to activate CD8⁺ T cell-mediated anti-tumor immunity,IRF3/IRF7-dependent type I interferon signaling is critical for DCactivation and cross-presentation of tumor antigens to promote CD8⁺ Tcell priming (Diamond et al. (2011) J. Exp. Med. 208(10):1989-2003;Fuertes et al. (2011) J. Exp. Med. 208(10):2005-2016). Type Iinterferons (IFN-α, IFN-β) are the signature cytokines induced by twodistinct TLR-dependent and TLR-independent signaling pathways. TheTLR-dependent pathway for inducing IFN-β occurs following endocytosis ofpathogens, whereby TLR3, 7, 8 and 9 detect pathogen-derived DNA and RNAelements within the endosomes. TLRs 7 and 8 recognize viral nucleosidesand nucleotides, and synthetic agonists of these, such as resiquimod andimiquimod have been clinically validated (Chi et al. (2017) Frontiers inPharmacology 8:304). Synthetic dsRNA, such as polyinosinic:polycytidylicacid (poly (I:C)) and poly ICLC, an analog that is formulated with polyL lysine to resist RNase digestion, is an agonist for TLR3 and MDA5pathways and a powerful inducer of IFN-β (Caskey et al. (2011) J. Exp.Med. 208(12):2357-66). TLR9 detection of endosomal CpG motifs present inviral and bacterial DNA can also induce IFN-β via IRF3. Additionally,TLR4 has been shown to induce IFN-β via MyD88-independent TRIFactivation of IRF3 (Owen et al. (2016) mBio.7:1 e02051-15). Itsubsequently was shown that TLR4 activation of DCs was independent oftype I IFN, so the ability of TLR4 to activate DCs via type I IFN is notlikely biologically relevant (Hu et al. (2015) Proc. Natl. Acad. Sci.U.S.A. 112:45). Further, TLR4 signaling has not been shown to directlyrecruit or activate CD8⁺ T cells.

Of the TLR-independent type I IFN pathways, one is mediated by hostrecognition of single-stranded (ss) and double-stranded (ds) RNA in thecytosol. These are sensed by RNA helicases, including retinoicacid-inducible gene I (RIG-I), melanoma differentiation-associated gene5 (MDA-5), and through the IFN-β promoter stimulator 1 (IPS-1; alsoknown as mitochondrial antiviral-signaling protein or MAVS) adaptorprotein-mediated phosphorylation of the IRF-3 transcription factor,leading to induction of IFN-β (Ireton and Gale (2011) Viruses3(6):906-919). Synthetic RIG-I-binding elements have also beendiscovered unintentionally in common lentiviral shRNA vectors, in theform of an AA dinucleotide sequence at the U6 promoter transcriptionstart site. Its subsequent deletion in the plasmid prevented confoundingoff-target type I IFN activation (Pebernard et al. (2004)Differentiation 72:103-111).

The second type of TLR-independent type I interferon induction pathwayis mediated through Stimulator of Interferon Genes (STING), a cytosolicER-resident adaptor protein that is now recognized as the centralmediator for sensing cytosolic dsDNA from infectious pathogens oraberrant host cell damage (Barber (2011) Immunol. Rev. 243(1):99-108).STING signaling activates the TANK binding kinase (TBK1)/IRF3 axis andthe NF-kB signaling axis, resulting in the induction of IFN-β and otherpro-inflammatory cytokines and chemokines that strongly activate innateand adaptive immunity (Burdette et al. (2011) Nature 478(7370):515-518).Sensing of cytosolic dsDNA through STING requires cyclic GMP-AMPsynthase (cGAS), a host cell nucleotidyl transferase that directly bindsdsDNA, and in response, synthesizes a cyclic dinucleotide (CDN) secondmessenger, cyclic GMP-AMP (cGAMP), which binds and activates STING (Sunet al. (2013) Science 339(6121):786-791; Wu et al. (2013) Science339(6121):826-830). CDNs derived from bacteria such as c-di-AMP producedfrom intracellular Listeria monocytogenes can also directly bind murineSTING, but only 3 of the 5 human STING alleles. Unlike the CDNs producedby bacteria, in which the two purine nucleosides are joined by aphosphate bridge with 3′-3′ linkages, the internucleotide phosphatebridge in the cGAMP synthesized by mammalian cGAS is joined by anon-canonical 2′-3′ linkage. These 2′-3′ molecules bind to STING with300-fold better affinity than bacterial 3′-3′ CDNs, and thus are morepotent physiological ligands of human STING (see, e.g., Civril et al.(2013) Nature 498(7454):332-337; Diner et al. (2013) Cell Rep.3(5):1355-1361; Gao et al. (2013) Sci. Signal 6(269):pl1; Ablasser etal. (2013) Nature 503(7477):530-534).

The cGAS/STING signaling pathway in humans may have evolved over time topreferentially respond to viral pathogens over bacterial pathogens, andthis can explain why bacterial vaccines harboring host tumor antigenshave made for poor CD8⁺ T cell priming vectors in humans.TLR-independent activation of CD8⁺ T cells by STING-dependent type I IFNsignaling from conventional DCs is the primary mechanism by whichviruses are detected, with TLR-dependent type I IFN production byplasmacytoid DCs operating only when the STING pathway has beenvirally-inactivated (Hervas-Stubbs et al. (2014) J. Immunol.193:1151-1161). Further, for bacteria such as S. typhimurium, whilecapable of inducing IFN-3 via TLR4, CD8⁺ T cells are neither induced norrequired for clearance or protective immunity (Lee et al. (2012)Immunol. Lett. 148(2): 138-143). The lack of physiologically relevantCD8⁺ T epitopes for many strains of bacteria, including S. typhimurium,has impeded both bacterial vaccine development and protective immunityto subsequent infections, even from the same genetic strains (Lo et al.(1999) J. Immunol. 162:5398-5406). Thus, bacterially-based cancerimmunotherapies are biologically limited in their ability to induce typeI IFN to recruit and activate CD8⁺ T cells, necessary to promote tumorantigen cross-presentation and durable anti-tumor immunity. Hence,engineering a bacterial immunotherapy provided herein to induceviral-like TLR-independent type I IFN signaling, rather thanTLR-dependent bacterial immune signaling, will preferentially induceCD8⁺ T cell mediated anti-tumor immunity.

STING activates innate immunity in response to sensing nucleic acids inthe cytosol. Downstream signaling is activated through binding of CDNs,which are synthesized by bacteria or by the host enzyme cGAS in responseto binding to cytosolic dsDNA. Bacterial and host-produced CDNs havedistinct phosphate bridge structures, which differentiates theircapacity to activate STING. IFN-3 is the signature cytokine of activatedSTING, and virally-induce type I IFN, rather than bacterially-inducedIFN, is required for effective CD8⁺ T cell mediated anti-tumor immunity.Immunostimulatory bacteria provided herein include those that are STINGagonists.

3. Salmonella Therapy

Salmonella is exemplary of a bacterial genus that can be used as acancer therapeutic. The Salmonella exemplified herein is an attenuatedspecies or one that, by virtue of the modifications for use as a cancertherapeutic, has reduced toxicity.

a. Tumor-Tropic Bacteria

A number of bacterial species have demonstrated preferential replicationwithin solid tumors when injected from a distal site. These include, butare not limited to, species of Salmonella, Bifodobacterium, Clostridium,and Escherichia. The natural tumor-homing properties of the bacteriacombined with the host's innate immune response to the bacterialinfection is thought to mediate the anti-tumor response. This tumortissue tropism has been shown to reduce the size of tumors to varyingdegrees. One contributing factor to the tumor tropism of these bacterialspecies is the ability to replicate in anoxic or hypoxic environments. Anumber of these naturally tumor-tropic bacteria have been furtherengineered to increase the potency of the antitumor response (reviewedin Zu et al. (2014) Crit. Rev. Microbiol. 40(3):225-235; and Felgner etal. (2017) Microbial Biotechnology 10(5):1074-1078).

b. Salmonella enterica Serovar typhimurium

Salmonella enterica Serovar typhimurium (S. typhimurium) is exemplary ofa bacterial species for use as an anti-cancer therapeutic. One approachto using bacteria to stimulate host immunity to cancer has been throughthe Gram-negative facultative anaerobe S. typhimurium, whichpreferentially accumulates in hypoxic and necrotic areas in the body,including tumor microenvironments. S. typhimurium accumulates in theseenvironments due to the availability of nutrients from tissue necrosis,the leaky tumor vasculature and their increased likelihood to survive inthe immune system-evading tumor microenvironment (Baban et al. (2010)Bioengineered Bugs 1(6):385-294). S. typhimurium is able to grow underboth aerobic and anaerobic conditions; therefore it is able to colonizesmall tumors that are less hypoxic and large tumors that are morehypoxic.

S. typhimurium is a Gram-negative, facultative pathogen that istransmitted via the fecal-oral route. It causes localizedgastrointestinal infections, but also enters the bloodstream andlymphatic system after oral ingestion, infecting systemic tissues suchas the liver, spleen and lungs. Systemic administration of wild-type S.typhimurium overstimulates TNF-α induction, leading to a cytokinecascade and septic shock, which, if left untreated, can be fatal. As aresult, pathogenic bacterial strains, such as S. typhimurium, must beattenuated to prevent systemic infection, without completely suppressingtheir ability to effectively colonize tumor tissues. Attenuation isoften achieved by mutating a cellular structure that can elicit animmune response, such as the bacterial outer membrane or limiting itsability to replicate in the absence of supplemental nutrients.

S. typhimurium is an intracellular pathogen that is rapidly taken up bymyeloid cells such as macrophages or it can induce its own uptake in innon-phagocytic cells such as epithelial cells. Once inside cells, it canreplicate within a Salmonella containing vacuole (SCV) and can alsoescape into the cytosol of some epithelial cells. Many of the moleculardeterminants of S. typhimurium pathogenicity have been identified andthe genes are clustered in Salmonella pathogenicity islands (SPIs). Thetwo best characterized pathogenicity islands are SPI-1 which isresponsible for mediating bacterial invasion of non-phagocytic cells,and SPI-2 which is required for replication within the SCV (Agbor andMcCormick (2011) Cell Microbiol. 13(12): 1858-1869). Both of thesepathogenicity islands encode macromolecular structures called type threesecretion systems (T3SS) that can translocate effector proteins acrossthe host membrane (Galan and Wolf-Watz (2006) Nature 444:567-573).

c. Bacterial Attenuation

Therapeutic bacteria for administration as a cancer treatment should beattenuated. Various methods for attenuation of bacterial pathogens areknown in the art. Auxotrophic mutations, for example, render bacteriaincapable of synthesizing an essential nutrient, and deletions/mutationsin genes such as aro, pur, gua, thy, nad and asd (U.S. PatentPublication No. 2012/0009153) are widely used. Nutrients produced by thebiosynthesis pathways involving these genes are often unavailable inhost cells, and as such, bacterial survival is challenging. For example,attenuation of Salmonella and other species can be achieved by deletionof the aroA gene, which is part of the shikimate pathway, connectingglycolysis to aromatic amino acid biosynthesis (Felgner et al. (2016)MBio 7(5):e01220-16). Deletion of aroA therefore results in bacterialauxotrophy for aromatic amino acids and subsequent attenuation (U.S.Patent Publication Nos. 2003/0170276, 2003/0175297, 2012/0009153,2016/0369282; International Application Publication Nos. WO 2015/032165and WO 2016/025582). Similarly, other enzymes involved in thebiosynthesis pathway for aromatic amino acids, including aroC and aroDhave been deleted to achieve attenuation (U.S. Patent Publication No.2016/0369282; International Patent Application Publication No. WO2016/025582). For example, S. typhimurium strain SL7207 is an aromaticamino acid auxotroph (aroA mutant); strains A1 and A1-R areleucine-arginine auxotrophs. VNP20009 is a purine auxotroph (purI⁻mutant). As shown herein, it is also auxotrophic for theimmunosuppressive nucleoside adenosine.

Mutations that attenuate bacteria also include, but are not limited to,mutations in genes that alter the biosynthesis of lipopolysaccharide,such as rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL,pagP, lpxR, arnT, eptA, and lpxT; mutations that introduce a suicidegene such as sacB, nuk, hok, gef kil or phlA; mutations that introduce abacterial lysis gene such as hly and cly; mutations in virulence factorssuch as IsyA, pag, prg, iscA, virG, plc and act; mutations that modifythe stress response such as recA, htrA, htpR, hsp and groEL; mutationsthat disrupt the cell cycle such as min; and mutations that disrupt orinactivate regulatory functions, such as cya, crp, phoP/phoQ, and ompR(U.S. Patent Publication Nos. 2012/0009153, 2003/0170276, 2007/0298012;U.S. Pat. No. 6,190,657; International Application Publication No. WO2015/032165; Felgner et al. (2016) Gut microbes 7(2):171-177; Broadwayet al. (2014) J. Biotechnology 192:177-178; Frahm et al. (2015) mBio6(2):e00254-15; Kong et al. (2011) Infection and Immunity79(12):5027-5038; Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419). Ideally, the genetic attenuations comprise gene deletionsrather than point mutations to prevent spontaneous compensatorymutations that might result in reversion to a virulent phenotype.

i. msbB⁻ Mutants

The enzyme lipid A biosynthesis myristoyltransferase, encoded by themsbB gene in S. typhimurium, catalyzes the addition of a terminalmyristyl group to the lipid A domain of lipopolysaccharide (LPS) (Low etal. (1999) Nat. Biotechnol. 17(1):37-41). Deletion of msbB thus altersthe acyl composition of the lipid A domain of LPS, the major componentof the outer membranes of Gram-negative bacteria. This modificationsignificantly reduces the ability of the LPS to induce septic shock,attenuating the bacterial strain and reducing the potentially harmfulproduction of TNFα, thus lowering systemic toxicity. S. typhimurium msbBmutants maintain their ability to preferentially colonize tumors overother tissues in mice and retain anti-tumor activity, thus increasingthe therapeutic index of Salmonella based immunotherapeutics (U.S.Patent Publication Nos. 2003/0170276, 2003/0109026, 2004/0229338,2005/0225088, 2007/0298012).

For example, deletion of msbB in the S. typhimurium strain VNP20009results in production of a predominantly penta-acylated LPS, which isless toxic than native hexa-acylated LPS and allows for systemicdelivery without the induction of toxic shock (Lee et al. (2000)International Journal of Toxicology 19:19-25). Other LPS mutations canbe introduced into the bacterial strains provided herein, including theSalmonella strains, that dramatically reduce virulence, and therebyprovide for lower toxicity, and permit administration of higher doses.

ii. purI⁻ Mutants

Immunostimulatory bacteria that can be attenuated by rendering themauxotrophic for one or more essential nutrients, such as purines (forexample, adenine), nucleosides (for example, adenosine) or amino acids(for example, arginine and leucine), are employed. In particular, inembodiments of the immunostimulatory bacteria provided herein, such asS. typhimurium, the bacteria are rendered auxotrophic for adenosine,which preferentially accumulates in tumor microenvironments. Hence,strains of immunostimulatory bacteria described herein are attenuatedbecause they require adenosine for growth, and they preferentiallycolonize TMEs, which, as discussed below, have an abundance ofadenosine.

Phosphoribosylaminoimidazole synthetase, an enzyme encoded by the purIgene (synonymous with the purM gene), is involved in the biosynthesispathway of purines. Disruption of the purI gene thus renders thebacteria auxotrophic for purines. In addition to being attenuated, purI⁻mutants are enriched in the tumor environment and have significantanti-tumor activity (Pawelek et al. (1997) Cancer Research57:4537-4544). It was previously described that this colonizationresults from the high concentration of purines present in theinterstitial fluid of tumors as a result of their rapid cellularturnover. Since the purI bacteria are unable to synthesize purines, theyrequire an external source of adenine, and it was thought that thiswould lead to their restricted growth in the purine-enriched tumormicroenvironment (Rosenberg et al. (2002) J. Immunotherapy25(3):218-225). While the VNP20009 strain was initially reported tocontain a deletion of the purI⁻ gene (Low et al. (2003) Methods inMolecular Medicine Vol. 90, Suicide Gene Therapy:47-59), subsequentanalysis of the entire genome of VNP20009 demonstrated that the purI⁻gene is not deleted, but is disrupted by a chromosomal inversion(Broadway et al. (2014) Journal of Biotechnology 192:177-178). Theentire gene is contained within two parts of the VNP20009 chromosomethat is flanked by insertion sequences (one of which has an activetransposase).

It is shown herein, that, purI mutant S. typhimurium strains areauxotrophic for the nucleoside adenosine, which is highly enriched intumor microenvironments. Hence, when using VNP20009, it is not necessaryto introduce any further modification to achieve adenosine auxotrophy.For other strains and bacteria, the purI gene can be disrupted as it hasbeen in VNP20009, or it can contain a deletion of all or a portion ofthe purI gene to prevent reversion to a wild-type gene.

iii. Combinations of Attenuating Mutations

A bacterium with multiple genetic attenuations by means of genedeletions on disparate regions of the chromosome is desirable forbacterial immunotherapies because the attenuation can be increased,while decreasing the possibility of reversion to a virulent phenotype byacquisition of genes by homologous recombination with a wild-typegenetic material. Restoration of virulence by homologous recombinationwould require two separate recombination events to occur within the sameorganism. Ideally the combinations of attenuating mutations selected foruse in an immunotherapeutic agent increases the tolerability withoutdecreasing the potency, thereby increasing the therapeutic index. Forexample, disruption of the msbB and purI genes in S. typhimurium strainVNP20009, has been used for tumor-targeting and growth suppression, andelicits low toxicity in animal models (Clairmont et al. (2000) J.Infect. Dis. 181:1996-2002; Bermudes et al. (2000) Cancer Gene Therapy:Past Achievements and Future Challenges, edited by Habib KluwerAcademic/Plenum Publishers, New York, pp. 57-63; Low et al. (2003)Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy:47-59; Leeet al. (2000) International Journal of Toxicology 19:19-25; Rosenberg etal. (2002) J. Immunotherapy 25(3):218-225; Broadway et al. (2014) J.Biotechnology 192:177-178; Loeffler et al. (2007) Proc. Natl. Acad. Sci.U.S.A. 104(31):12879-12883; Luo et al. (2002) Oncology Research12:501-508). When VNP20009 (msbB⁻/purI⁻) was administered to micebearing syngeneic or human xenograft tumors, the bacteria accumulatedpreferentially within the extracellular components of tumors at ratiosexceeding 300-1000 to 1, reduced TNFα induction, and demonstrated tumorregression and prolonged survival compared to control mice (Clairmont etal. (2000) J. Infect. Dis. 181:1996-2002). Results from the Phase 1clinical trial in humans, however, revealed that while VNP20009 wasrelatively safe and well tolerated, poor accumulation was observed inhuman melanoma tumors, and very little anti-tumor activity wasdemonstrated (Toso et al. (2002) J. Clin. Oncol. 20(1): 142-152). Higherdoses, which are required to manifest any anti-tumor activity, were notpossible due to toxicity.

Thus, further improvements are needed. The immunostimulatory bacteriaprovided herein address this problem.

iv. VNP20009 and Other Attenuated and Wild-Type S. typhimurium Strains

The starting strain can be a wild-type non-attenuated strain, such as astrain having all of the identifying characteristics of ATCC 14028. Thestrain is then modified to increase its specificity or targeting to thetumor microenvironment or to tumor cells and/or to tumor resident immunecells. It also can be modified to be auxotrophic for adenosine. Thestrain can be rendered flagellin⁻ (fliC⁻/fljB⁻), and optionally one ormore of msbB⁻, purI⁻/M⁻, and pagP⁻. The strains also can be asd⁻. Themodified strains encode a therapeutic product on a plasmid, generallypresent in low to medium copy number, under control of a promoterrecognized by a mammalian host, such as RNA polymerase II or III.Additional regulatory sequences to control expression in the tumormicroenvironment and trafficking in the cells also can be included.

Exemplary of a therapeutic bacterium that can be modified as describedherein is the strain designated as VNP20009 (ATCC #202165, YS1646),which is derived from the strain ATCC accession no. 14028. The straindesignated VNP20009 (ATCC #202165, YS1646), was a clinical candidate,and at least 50,000-fold attenuated for safety by deletion of the msbBand purI⁻ genes (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002;Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide GeneTherapy:47-59; Lee et al. (2000) International Journal of Toxicology19:19-25). Similar strains of Salmonella that are attenuated also arecontemplated. As described above, deletion of msbB alters thecomposition of the lipid A domain of lipopolysaccharide, the majorcomponent of Gram-negative bacterial outer membranes (Low et al. (1999)Nat. Biotechnol. 17(1):37-41). This prevents lipopolysaccharide-inducedseptic shock, attenuating the bacterial strain and lowering systemictoxicity, while reducing the potentially harmful production of TNFα(Dinarello, C. A. (1997) Chest 112(6 Suppl):321S-329S; Low et al. (1999)Nat. Biotechnol. 17(1):37-41). Deletion of the purI⁻ gene renders thebacteria auxotrophic for purines, which further attenuates the bacteriaand enriches it in the tumor microenvironment (Pawelek et al. (1997)Cancer Res. 57:4537-4544; Broadway et al. (2014) J. Biotechnology192:177-178).

Accumulation of VNP20009 in tumors results from a combination of factorsincluding: the inherent invasiveness of the parental strain, ATCCaccession number 14028, its ability to replicate in hypoxicenvironments, and its requirement for high concentrations of purinesthat are present in the interstitial fluid of tumors. It is shown hereinthat VNP20009 also is auxotrophic for the nucleoside adenosine, whichcan accumulate to pathologically high levels in the tumormicroenvironment and contribute to an immunosuppressive tumormicroenvironment (Peter Vaupel and Arnulf Mayer Oxygen Transport toTissue XXXVII, Advances in Experimental Medicine and Biology 876 chapter22, pp. 177-183). VNP20009 was administered into mice bearing syngeneicor human xenograft tumors, the bacteria accumulated preferentiallywithin the extracellular components of tumors at ratios exceeding300-1000 to 1 and demonstrated tumor growth inhibition as well asprolonged survival compared to control mice (Clairmont et al. (2000) J.Infect. Dis. 181:1996-2002). Results from the Phase 1 clinical trialrevealed that while VNP20009 was relatively safe and well tolerated,poor accumulation was observed in human melanoma tumors, and very littleanti-tumor activity was demonstrated (Toso et al. (2002) J. Clin. Oncol.20(1):142-152). Higher doses, which would be required to affect anyanti-tumor activity, were not possible due to toxicity that correlatedwith high levels of pro-inflammatory cytokines. The modificationsprovided herein, including the flagellin deletion (fliC⁻/fljB⁻), andoptional pagP⁻ and/or hilA⁻ modifications, significantly increaseaccumulation of the immunostimulatory bacteria in tumors, in the tumormicroenvironment and/or in tumor-resident immune cells, such as myeloidcells. Other modifications that increase targeting to immune cells, andeliminate infection of other cells, such as epithelial cells, increasethe accumulation of the bacteria in the tumors and in the tumormicroenvironment. Additional modifications to render the wild-typebacteria auxotrophic for adenosine further increases accumulation in thetumor microenvironment.

Other strains of S. typhimurium can be used for tumor-targeted deliveryof therapeutic proteins and therapy, such as, for example,leucine-arginine auxotroph A-1 (Zhao et al. (2005) Proc. Natl. Acad.Sci. USA 102(3):755-760; Yu et al. (2012) Scientific Reports 2:436; U.S.Pat. No. 8,822,194; U.S. Patent Publication No. 2014/0178341) and itsderivative AR-1 (Yu et al. (2012) Scientific Reports 2:436; Kawagushi etal. (2017) Oncotarget 8(12): 19065-19073; Zhao et al. (2006) Cancer Res.66(15):7647-7652; Zhao et al. (2012) Cell Cycle 11(1):187-193; Tome etal. (2013) Anticancer Research 33:97-102; Murakami et al. (2017)Oncotarget 8(5):8035-8042; Liu et al. (2016) Oncotarget7(16):22873-22882; Binder et al. (2013) Cancer Immunol Res.1(2):123-133); aroA⁻ mutant S. typhimurium strain SL7207 (Guo et al.(2011) Gene therapy 18:95-105; U.S. Patent Publication Nos.2012/0009153, 2016/0369282 and 2016/0184456) and its obligate anaerobederivative YB1 (WO 2015/032165; Yu et al. (2012) Scientific Reports2:436; Leschner et al. (2009) PLoS ONE 4(8): e6692; Yu et al. (2012)Scientific Reports 2:436); aroA⁻/aroD⁻ mutant S. typhimurium strainBRD509, a derivative of the SL1344 (WT) strain (Yoon et al. (2017)European J. of Cancer 70:48-61); asd⁻/cya-/crp mutant S. typhimuriumstrain χ4550 (Sorenson et al. (2010) Biology: Targets & Therapy 4:61-73)and phoP⁻/phoQ⁻ S. typhimurium strain LH430 (International ApplicationPublication No. WO 2008/091375).

The strain VNP20009 failed to show a clinical benefit in a studyinvolving patients with advanced melanoma, but the treatment was safelyadministered to advanced cancer patients. A maximum tolerated dose (MTD)was established. Hence, this strain, as well as other similarlyengineered bacterial strains, can be used as a starting material fortumor-targeting, therapeutic delivery vehicles. Modifications providedherein provide a strategy to increase efficacy, by increasing theanti-tumor efficiency and/or the safety and tolerability of thetherapeutic agent.

v. S. typhimurium Engineered To Deliver Macromolecules

S. typhimurium also has been modified to deliver the tumor-associatedantigen (TAA) survivin (SVN) to APCs to prime adaptive immunity (U.S.Patent Publication No. 2014/0186401; Xu et al. (2014) Cancer Res.74(21):6260-6270). SVN is an inhibitor of apoptosis protein (IAP) whichprolongs cell survival and provides cell cycle control, and isoverexpressed in all solid tumors and poorly expressed in normaltissues. This technology utilizes Salmonella Pathogenicity Island 2(SPI-2) and its type III secretion system (T3SS) to deliver the TAAsinto the cytosol of APCs, which then are activated to induceTAA-specific CD8⁺ T cells and anti-tumor immunity (Xu et al. (2014)Cancer Res. 74(21):6260-6270). Similar to the Listeria-based TAAvaccines, this approach has shown promise in mouse models, but has yetto demonstrate effective tumor antigen-specific T cell priming inhumans.

In addition to gene delivery, S. typhimurium also has been used for thedelivery of small interfering RNAs (siRNAs) and short hairpin RNAs(shRNAs) for cancer therapy. For example, attenuated S. typhimurium havebeen modified to express certain shRNAs, such as those that target STAT3and IDO1 (PCT/US2007/074272, and U.S. Pat. No. 9,453,227). VNP20009transformed with an shRNA plasmid against the immunosuppressive geneindolamine deoxygenase (IDO), successfully silenced IDO expression in amurine melanoma model, resulting in tumor cell death and significanttumor infiltration by neutrophils (Blache et al. (2012) Cancer Res.72(24):6447-6456). Combining this vector with the co-administration of ahyaluronidase, such as PEGylated soluble PH20 (PEGPH20; an enzyme thatdepletes extracellular hyaluronan), shows positive results in thetreatment of pancreatic ductal adenocarcinoma tumors (Manuel et al.(2015) Cancer Immunol. Res. 3(9): 1096-1107; U.S. Patent Publication No.2016/0184456). In another study, an S. typhimurium strain attenuated bya phoP/phoQ deletion and expressing a signal transducer and activator oftranscription 3 (STAT3)-specific shRNA, was found to inhibit tumorgrowth and reduce the number of metastatic organs, extending the life ofC57BL6 mice (Zhang et al. (2007) Cancer Res. 67(12):5859-5864). Inanother example, S. typhimurium strain SL7207 has been used for thedelivery of shRNA targeting CTNNB1, the gene that encodes β-catenin (Guoet al. (2011) Gene therapy 18:95-105; U.S. Patent Publication Nos.2009/0123426, 2016/0369282), while S. typhimurium strain VNP20009 hasbeen used in the delivery of shRNA targeting the STAT3 (Manuel et al.(2011) Cancer Res. 71(12):4183-4191; U.S. Patent Publication Nos.2009/0208534, 2014/0186401, 2016/0184456; International ApplicationPublication Nos. WO 2008/091375, WO 2012/149364). siRNAs targeting theautophagy genes Atg5 and Beclinl have been delivered to tumor cellsusing S. typhimurium strains A1-R and VNP20009 (Liu et al. (2016)Oncotarget 7(16):22873-22882). Improvement of such strains is needed sothat they more effectively colonize tumors, the TME, and/ortumor-resident immune cells, and also stimulate the immune response, andhave other advantageous properties, such as the immunostimulatorybacteria provided herein. Modifications of various bacteria have beendescribed in International PCT Application Publication No. WO2019/014398 and U.S. Publication No. 2019/0017050 A1. The bacteriadescribed in each of these publications, also described herein, can bemodified as described herein to further improve the immunostimulatoryand tumor-targeting properties.

The bacteria can be modified as described herein to have reducedinflammatory effects, and thus, to be less toxic. As a result, forexample, higher dosages can be administered. Any of these strains ofSalmonella, as well as other species of bacteria, known to those ofskill in the art and/or listed above and herein, can be modified asdescribed herein, such as by introducing adenosine auxotrophy a plasmidencoding a therapeutic product, such as an immunostimulatory proteinand/or RNAi, such as miRNA or shRNA, or an antibody or fragment thereof,for inhibiting an immune checkpoint, and other modifications asdescribed herein. Exemplary are the S. typhimurium species describedherein.

The bacterial strains provided herein are engineered to delivertherapeutic molecules. The strains herein deliver immunostimulatoryproteins, such as cytokines, that promote an anti-tumor immune responsein the tumor microenvironment. The strains also can include genomicmodifications that reduce pyroptosis of phagocytic cells, therebyproviding for a more robust immune response, and/or reduce or eliminatethe ability to infect/invade epithelial cells, but retain the ability toinfect/invade phagocytic cells, so that they accumulate more effectivelyin tumors and in tumor-resident immune cells. The bacterial strains alsocan be modified to encode therapeutic products, including, for example,RNAi targeted and inhibitory to immune checkpoints, and also to othersuch targets.

4. Enhancements of Immunostimulatory Bacteria to Increase TherapeuticIndex

Provided herein are enhancements to immunostimulatory bacteria thatreduce toxicity and improve the anti-tumor activity. Exemplary of suchenhancements are the following. They are described with respect toSalmonella, particularly S. typhimurium; it is understood that theskilled person can effect similar enhancements in other bacterialspecies and other Salmonella strains.

a. asd Gene Deletion

The asd gene in bacteria encodes an aspartate-semialdehydedehydrogenase. asd- mutants of S. typhimurium have an obligaterequirement for diaminopimelic acid (DAP) which is required for cellwall synthesis and will undergo lysis in environments deprived of DAP.This DAP auxotrophy can be used for plasmid selection and maintenance ofplasmid stability in vivo without the use of antibiotics when the asdgene is complemented in trans on a plasmid. Non-antibiotic-based plasmidselection systems are advantageous and allow for 1) use of administeredantibiotics as rapid clearance mechanism in the event of adversesymptoms, and 2) for antibiotic-free scale up of production, where suchuse is commonly avoided. The asd gene complementation system providesfor such selection (Galan et al. (1990) Gene 28:29-35). The use of theasd gene complementation system to maintain plasmids in the tumormicroenvironment is expected to increase the potency of S. typhimuriumengineered to deliver plasmids encoding genes or interfering RNAs.

An alternative use for an asd mutant of S. typhimurium is to exploit theDAP auxotrophy to produce an autolytic (or suicidal) strain for deliveryof macromolecules to infected cells without the ability to persistentlycolonize host tumors. Deletion of the asd gene makes the bacteriaauxotrophic for DAP when grown in vitro or in vivo. An example describedherein, provides an asd deletion strain that is auxotrophic for DAP andcontains a plasmid suitable for delivery of RNAi, such as shRNA ormi-RNA, that does not contain an asd complementing gene, resulting in astrain that is defective for replication in vivo. This strain ispropagated in vitro in the presence of DAP and grows normally, and thenis administered as an immunotherapeutic agent to a mammalian host whereDAP is not present. The suicidal strain is able to invade host cells butis not be able to replicate due to the absence of DAP in mammaliantissues, lysing automatically and delivering its cytosolic contents(e.g., plasmids or proteins). In examples provided herein, an asd genedeleted strain of VNP20009 was further modified to express an LLOprotein lacking its endogenous periplasmic secretion signal sequence,causing it to accumulate in the cytoplasm of the Salmonella. LLO is acholesterol-dependent pore forming hemolysin from Listeria monocytogenesthat mediates phagosomal escape of bacteria. When the autolytic strainis introduced into tumor bearing mice, the bacteria are taken up byphagocytic immune cells and enter the Salmonella containing vacuole(SCV). In this environment, the lack of DAP will prevent bacterialreplication, and result in autolysis of the bacteria in the SCV. Lysisof the suicidal strain will then allow for release of the plasmid andthe accumulated LLO that will form pores in the cholesterol-containingSVC membrane, and allow for delivery of the plasmid into the cytosol ofthe host cell.

b. Adenosine Auxotrophy

Metabolites derived from the tryptophan and ATP/adenosine pathways aremajor drivers in forming an immunosuppressive environment within thetumor. Adenosine, which exists in the free form inside and outside ofcells, is an effector of immune function. Adenosine decreases T-cellreceptor induced activation of NF-κB, and inhibits IL-2, IL-4, andIFN-γ. Adenosine decreases T-cell cytotoxicity, increases T-cell anergy,and increases T-cell differentiation to Foxp3⁺ or Lag-3⁺ regulatory(T-reg) T-cells. On NK cells, adenosine decreases IFN-γ production, andsuppresses NK cell cytotoxicity. Adenosine blocks neutrophil adhesionand extravasation, decreases phagocytosis, and attenuates levels ofsuperoxide and nitric oxide. Adenosine also decreases the expression ofTNF-α, IL-12, and MIP-1a on macrophages, attenuates MHC Class IIexpression, and increases levels of IL-10 and IL-6. Adenosineimmunomodulation activity occurs after its release into theextracellular space of the tumor and activation of adenosine receptors(ADRs) on the surface of target immune cells, cancer cells orendothelial cells. The high adenosine levels in the tumormicroenvironment result in local immunosuppression, which limits thecapacity of the immune system to eliminate cancer cells.

Extracellular adenosine is produced by the sequential activities ofmembrane associated ectoenzymes, CD39 and CD73, which are expressed ontumor stromal cells, together producing adenosine by phosphohydrolysisof ATP or ADP produced from dead or dying cells. CD39 convertsextracellular ATP (or ADP) to 5′AMP, which is converted to adenosine by5′AMP. Expression of CD39 and CD73 on endothelial cells is increasedunder the hypoxic conditions of the tumor microenvironment, therebyincreasing levels of adenosine. Tumor hypoxia can result from inadequateblood supply and disorganized tumor vasculature, impairing delivery ofoxygen (Carroll and Ashcroft (2005) Expert. Rev. Mol. Med. 7(6): 1-16).Hypoxia, which occurs in the tumor microenvironment, also inhibitsadenylate kinase (AK), which converts adenosine to AMP, leading to veryhigh extracellular adenosine concentrations. The extracellularconcentration of adenosine in the hypoxic tumor microenvironment hasbeen measured at 10-100 μM, which is up to about 100-1000 fold higherthan the typical extracellular adenosine concentration of approximately0.1 μM (Vaupel et al. (2016) Adv. Exp. Med. Biol. 876:177-183; Antonioliet al. (2013) Nat. Rev. Can. 13:842-857). Since hypoxic regions intumors are distal from microvessels, the local concentration ofadenosine in some regions of the tumor can be higher than others.

To direct effects to inhibit the immune system, adenosine also cancontrol cancer cell growth and dissemination by effects on cancer cellproliferation, apoptosis and angiogenesis. For example, adenosine canpromote angiogenesis, primarily through the stimulation of A_(2A) andA_(2B) receptors. Stimulation of the receptors on endothelial cells canregulate the expression of intercellular adhesion molecule 1 (ICAM-1)and E-selectin on endothelial cells, maintain vascular integrity, andpromote vessel growth (Antonioli et al. (2013) Nat. Rev. Can.13:842-857). Activation of one or more of A_(2A), A_(2B) or A₃ onvarious cells by adenosine can stimulate the production of thepro-angiogenic factors, such as vascular endothelial growth factor(VEGF), interleukin-8 (IL-8) or angiopoietin 2 (Antonioli et al. (2013)Nat. Rev. Can. 13:842-857).

Adenosine also can directly regulate tumor cell proliferation, apoptosisand metastasis through interaction with receptors on cancer cells. Forexample, studies have shown that the activation of A₁ and A₂Areceptorspromote tumor cell proliferation in some breast cancer cell lines, andactivation of A_(2B) receptors have cancer growth-promoting propertiesin colon carcinoma cells (Antonioli et al. (2013) Nat. Rev. Can.13:842-857). Adenosine also can trigger apoptosis of cancer cells, andvarious studies have correlated this activity to activation of theextrinsic apoptotic pathway through A₃ or the intrinsic apoptoticpathway through A_(2A) and A_(2B) (Antonioli et al. (2013)). Adenosinecan promote tumor cell migration and metastasis, by increasing cellmotility, adhesion to the extracellular matrix, and expression of cellattachment proteins and receptors to promote cell movement and motility.

The extracellular release of adenosine triphosphate (ATP) occurs fromstimulated immune cells and damaged, dying or stressed cells. The NLRfamily pyrin domain-containing 3 (NLRP3) inflammasome, when stimulatedby this extracellular release of ATP, activates caspase-1 and results inthe secretion of the cytokines IL-1β and IL-18, which in turn activateinnate and adaptive immune responses (Stagg and Smyth (2010) Oncogene29:5346-5358). ATP is catabolized into adenosine by the enzymes CD39 andCD73. Activated adenosine acts as a highly immunosuppressive metabolitevia a negative-feedback mechanism and has a pleiotropic effect againstmultiple immune cell types in the hypoxic tumor microenvironment (Staggand Smyth (2010) Oncogene 29:5346-5358). Adenosine receptors A_(2A) andA_(2B) are expressed on a variety of immune cells and are stimulated byadenosine to promote cAMP-mediated signaling changes, resulting inimmunosuppressive phenotypes of T-cells, B-cells, NK cells, dendriticcells, mast cells, macrophages, neutrophils, and NKT cells. As a resultof this, adenosine levels can accumulate to over one hundred times theirnormal concentration in pathological tissues, such as solid tumors,which have been shown to overexpress ecto-nucleotidases, such as CD73.Adenosine has also been shown to promote tumor angiogenesis anddevelopment. An engineered bacterium that is auxotrophic for adenosinewould thus exhibit enhanced tumor-targeting and colonization.

Immunostimulatory bacteria, such as Salmonella typhi, can be madeauxotrophic for adenosine by deletion of the tsx gene (Bucarey et al.(2005) Infection and Immunity 73(10):6210-6219) or by deletion of purD(Husseiny (2005) Infection and Immunity 73(3):1598-1605). In the Gramnegative bacteria Xanthomonas oryzae, apurD gene knockout was shown tobe auxotrophic for adenosine (Park et al. (2007) FEMS Microbiol. Lett.276:55-59). As exemplified herein, S. typhimurium strain VNP20009, isauxotrophic for adenosine due to its purI deletion, hence, furthermodification to render it auxotrophic for adenosine is not required.Hence, embodiments of the immunostimulatory bacterial strains, asprovided herein, are auxotrophic for adenosine. Such auxotrophicbacteria selectively replicate in the tumor microenvironment, furtherincreasing accumulation and replication of the administered bacteria intumors and decreasing the levels of adenosine in and around tumors,thereby reducing or eliminating the immunosuppression caused byaccumulation of adenosine. Exemplary of such bacteria, provided hereinis a modified strain of S. typhimurium containing purI⁻/msbB⁻ mutationsto provide adenosine auxotrophy.

c. Flagellin Deficient Strains

Flagella are organelles on the surface of bacteria that are composed ofa long filament attached via a hook to a rotary motor that can rotate ina clockwise or counterclockwise manner to provide a means forlocomotion. Flagella in S. typhimurium are important for chemotaxis andfor establishing an infection via the oral route, due to the ability tomediate motility across the mucous layer in the gastrointestinal tract.While flagella have been demonstrated to be required for chemotaxis toand colonization of tumor cylindroids in vitro (Kasinskas and Forbes(2007) Cancer Res. 67(7):3201-3209), and motility has been shown to beimportant for tumor penetration (Toley and Forbes (2012) Integr. Biol.(Camb). 4(2): 165-176), flagella are not required for tumor colonizationin animals when the bacteria are administered intravenously (Stritzkeret al. (2010) International Journal of Medical Microbiology300:449-456). Each flagellar filament is composed of tens of thousandsof flagellin subunits. The S. typhimurium chromosome contains two genes,fliC and fljB, that encode antigenically distinct flagellin monomers.Mutants defective for both fliC and fljB are nonmotile and avirulentwhen administered via the oral route of infection, but maintainvirulence when administered parenterally.

Flagellin is a major pro-inflammatory determinant of Salmonella (Zeng etal. (2003) J Immunol 171:3668-3674), and is directly recognized by TLR5on the surface of cells, and by NLCR4 in the cytosol (Lightfield et al.(2008) Nat. Immunol. 9(10): 1171-1178). Both pathways lead topro-inflammatory responses resulting in the secretion of cytokines,including IL-1β, IL-18, TNF-α and IL-6. Attempts have been made to makeSalmonella-based cancer immunotherapy more potent by increasing thepro-inflammatory response to flagellin by engineering the bacteria tosecrete Vibrio vulnificus flagellin B, which induces greaterinflammation than flagellin encoded by fliC and fljB (Zheng et al.(2017) Sci. Transl. Med. 9(376):eaak9537).

Herein, Salmonella bacteria, S. typhimurium, are engineered to lack bothflagellin subunits fliC and fljB, to reduce pro-inflammatory signaling.For example, as shown herein, a Salmonella strain lacking msbB, whichresults in reduced TNF-alpha induction, is combined with fliC and fljBknockouts. This results in a Salmonella strain that has a combinedreduction in TNF-alpha induction and reduction in TLR5 recognition.These modifications can be combined with msbB⁻, fliC⁻ and fljB⁻, andtransformed with an immunostimulatory plasmid, optionally containingCpGs, and a therapeutic molecule, such as an antibody or RNAimolecule(s) targeting an immune checkpoint, such as TREX1, PD-L1, VISTA,SIRP-alpha, TGF-beta, beta-catenin, CD47, VEGF, and combinationsthereof. The resulting bacteria have reduced pro-inflammatory signaling,but robust anti-tumor activity.

For example, as provided herein, a fliC and fljB double mutant wasconstructed in the asd deleted strain of S. typhimurium VNP20009.VNP20009, which is attenuated for virulence by disruption of purI/purM,was also engineered to contain an msbB deletion that results inproduction of a lipid A subunit that is less toxigenic than wild-typelipid A. This results in reduced TNF-α production in the mouse modelafter intravenous administration, compared to strains with wild-typelipid A. The resulting strain is exemplary of strains that areattenuated for bacterial inflammation by modification of lipid A toreduce TLR2/4 signaling, and deletion of the flagellin subunits toreduce TLR5 recognition and inflammasome induction. Deletion of theflagellin subunits combined with modification of the LPS allows forgreater tolerability in the host, and directs the immuno-stimulatoryresponse towards delivery of RNA interference against desired targets inthe TME which elicit an anti-tumor response and promote an adaptiveimmune response to the tumor.

d. Salmonella Engineered to Escape the Salmonella Containing Vacuole(SCV)

Salmonella, such as S. typhimurium, are intracellular pathogens thatreplicate primarily in a membrane bound compartment called a Salmonellacontaining vacuole (SCV). In some epithelial cell lines and at a lowfrequency, S. typhimurium have been shown to escape into the cytosolwhere they can replicate. Salmonella engineered to escape the SCV withhigher efficiency will be more efficient at delivering macromolecules,such as plasmids, as the lipid bilayer of the SCV is a potentialbarrier. Provided herein are Salmonella and methods that have enhancedfrequency of SCV escape. This is achieved by deletion of genes requiredfor Salmonella induced filament (SIF) formation. These mutants have anincreased frequency of SCV escape and can replicate in the cytosol.

For example, enhanced plasmid delivery using a sifA mutant of S.typhimurium has been demonstrated. The sifA gene encodes SPI-2, T3SS-2secreted effector protein that mimics or activates a RhoA family of hostGTPases (Ohlson et al. (2008) Cell Host & Microbe 4:434-446). Othergenes encoding secreted effectors involved in SIF formation can betargeted. These include, for example, sseJ, sseL, sopD2, pipB2, sseF,sseG, spvB, and steA. Enhancing the escape of S. typhimurium byprevention of SIF formation releases live bacteria into the cytosol,where they can replicate.

Another method to enhance S. typhimurium escape from the SCV andincrease the delivery of macromolecules such as plasmids, is theexpression of a heterologous hemolysin that results in pore formationin, or rupture of, the SCV membrane. One such hemolysin is theListeriolysin O protein (LLO) from Listeria monocytogenes, which isencoded by the hlyA gene. LLO is a cholesterol-dependent pore-formingcytolysin that is secreted from L. monocytogenes and is primarilyresponsible for phagosomal escape and entry into the cytosol of hostcells. Secretion of LLO from S. typhimurium can result in bacterialescape and lead to replication in the cytosol. To prevent intact S.typhimurium from escaping the SCV and replicating in the cytosol, thenucleotides encoding the signal sequence can be removed from the gene.In this manner, the active LLO is contained within the cytoplasm of theS. typhimurium and LLO is only released when the bacteria undergo lysis.As provided herein, VNP20009 engineered to express cytoLLO to enhancedelivery of plasmids for expression of interfering RNAs to targets, suchas TREX1, can increase the therapeutic potency of the immunostimulatorybacteria.

e. Deletions in Salmonella Genes Required for Biofilm Formation

Bacteria and fungi are capable of forming multicellular structurescalled biofilms. Bacterial biofilms are encased within a mixture ofsecreted and cell wall-associated polysaccharides, glycoproteins, andglycolipids, as well as extracellular DNA, known collectively asextracellular polymeric substances. These extracellular polymericsubstances protect the bacteria from multiple insults, such as cleaningagents, antibiotics, and antimicrobial peptides. Bacterial biofilmsallow for colonization of surfaces, and are a cause of significantinfection of prosthetics, such as injection ports and catheters.Biofilms can also form in tissues during the course of an infection,which leads to increases in the duration of bacterial persistence andshedding, and limits the effectiveness of antibiotic therapies. Chronicpersistence of bacteria in biofilms is associated with increasedtumorigenesis, for example in S. typhi infection of the gall bladder (DiDomenico et al. (2017) Int. J. Mol. Sci. 18:1887).

S. typhimurium biofilm formation is regulated by CsgD. CsgD activatesthe csgBAC operon, which results in increased production of the curlifimbrial subunits CsgA and CsgB (Zakikhani et al. (2010) MolecularMicrobiology 77(3):771-786). CsgA is recognized as a PAMP by TLR2 andinduces production of IL-8 from human macrophages (Tukel et al. (2005)Molecular Microbiology 58(1):289-304). Further, CsgD indirectlyincreases cellulose production by activating the adrA gene that encodesfor di-guanylate cyclase. The small molecule cyclic di-guanosinemonophosphate (c-di-GMP) generated by AdrA is a ubiquitous secondarymessenger found in almost all bacterial species. The AdrA-mediatedincrease in c-di-GMP enhances expression of the cellulose synthetasegene bcsA, which in turn increases cellulose production via stimulationof the bcsABZC and bcsEFG operons. Reduction in the capability ofimmunostimulatory bacteria such as S. typhimurium to form biofilms canbe achieved through deletion of genes involved in biofilm formation suchas, for example, csgD, csgA, csgB, adrA, bcsA, bcsB, bcsZ, bcsE, bcsF,bcsG, dsbA, or dsbB (Anwar et al. (2014) PLoS ONE 9(8):e106095).

S. typhimurium can form biofilms in solid tumors as protection againstphagocytosis by host immune cells. Salmonella mutants that cannot formbiofilms are taken up more rapidly by host phagocytic cells and arecleared from infected tumors (Crull et al. (2011) Cellular Microbiology13(8):1223-1233). This increase in intracellular localization withinphagocytic cells can reduce the persistence of extracellular bacteria,and enhance the effectiveness of plasmid delivery and gene knockdown byRNA interference as described herein. Immunostimulatory bacteriaengineered to reduce biofilm formation, will increase clearance ratefrom tumors/tissues and therefore increase the tolerability of thetherapy, and will prevent colonization of prosthetics in patients,thereby increasing the therapeutic benefit of these strains. Adenosinemimetics can inhibit S. typhimurium biofilm formation, indicating thatthe high adenosine concentration in the tumor microenvironment cancontribute to tumor-associated biofilm formation (Koopman et al. (2015)Antimicrob. Agents Chemother. 59:76-84). As provided herein, liveattenuated strains of bacteria, such as S. typhimurium, that contain apurI disruption (and therefore, colonize adenosine-rich tumors), and arealso prevented from forming biofilms, by deletion of one or more genesrequired for biofilm formation, are engineered to deliver plasmidsencoding interfering RNA to stimulate a robust anti-tumor immuneresponse.

The adrA gene encodes a di-guanylate cyclase that produces c-di-GMP,which is required for S. typhimurium biofilm formation. c-di-GMP bindsto and is an agonist for the host cytosolic protein STING. As describedabove, STING agonists are pursued as anti-cancer treatments, vaccineadjuvants, and bacteria engineered to secrete cyclic di-nucleotides foruse in immunotherapies (Libanova 2012, Synlogic 2018 AACR poster).Immunostimulatory bacteria that are reduced in c-di-GMP production viathe deletion of adrA is counterintuitive, but bacterial mutants, such asS. typhimurium mutants, that are unable to form biofilms (including anadrA mutant), have demonstrated reduced therapeutic potential in mousetumor models (Crull et al. (2011) Cellular Microbiology13(8):1223-1233). Several human alleles of STING are refractory tobinding bacterially-produced 3′3′ CDNs (Corrales et al. (2015) CellReports 11:1022-1023).

As described herein, bacterial strains, such as S. typhimurium strains,that are engineered to be adenosine auxotrophic, and are reduced intheir ability to induce pro-inflammatory cytokines by modification ofthe LPS and/or deletion of flagellin, and/or deletion of genes requiredfor biofilm formation, and further modified to deliver interfering RNAs,promote robust anti-tumor immune responses.

f. Deletions in Genes in the LPS Biosynthetic Pathway

The LPS of Gram negative bacteria is the major component of the outerleaflet of the bacterial membrane. It is composed of three major parts,lipid A, a non-repeating core oligosaccharide, and the O antigen (or Opolysaccharide). O antigen is the outermost portion on LPS and serves asa protective layer against bacterial permeability, however, the sugarcomposition of O antigen varies widely between strains. The lipid A andcore oligosaccharide vary less, and are more typically conserved withinstrains of the same species. Lipid A is the portion of LPS that containsendotoxin activity. It is typically a disaccharide decorated withmultiple fatty acids. These hydrophobic fatty acid chains anchor the LPSinto the bacterial membrane, and the rest of the LPS projects from thecell surface. The lipid A domain is responsible for much of the toxicityof Gram-negative bacteria. Typically, LPS in the blood is recognized asa significant pathogen associated molecular pattern (PAMP) and induces aprofound pro-inflammatory response. LPS is the ligand for amembrane-bound receptor complex comprising CD14, MD2 and TLR4. TLR4 is atransmembrane protein that can signal through the MyD88 and TRIFpathways to stimulate the NFκB pathway and result in the production ofpro-inflammatory cytokines such as TNF-α and IL-1β, the result of whichcan be endotoxic shock, which can be fatal. LPS in the cytosol ofmammalian cells can bind directly to the CARD domains of caspases 4, 5,and 11, leading to autoactivation and pyroptotic cell death (Hagar etal. (2015) Cell Research 25:149-150). The composition of lipid A and thetoxigenicity of lipid A variants is well documented. For example, amonophosphorylated lipid A is much less inflammatory than lipid A withmultiple phosphate groups. The number and length of the of acyl chainson lipid A can also have a profound impact on the degree of toxicity.Canonical lipid A from E. coli has six acyl chains, and thishexa-acylation is potently toxic. S. typhimurium lipid A is similar tothat of E. coli; it is a glucosamine disaccharide that carries fourprimary and two secondary hydroxyacyl chains (Raetz and Whitfield (2002)Annu. Rev. Biochem. 71:635-700).

As described above, msbB mutants of S. typhimurium cannot undergo theterminal myristoylation of its LPS and produce predominantlypenta-acylated LPS that is significantly less toxic than hexa-acylatedlipid A. The modification of lipid A with palmitate is catalyzed bypalmitoyl transferase (PagP). Transcription of the pagP gene is undercontrol of the PhoP/PhoQ system which is activated by low concentrationsof magnesium, e.g., inside the SCV. Thus, the acyl content of S.typhimurium is variable, and with wild-type bacteria it can be hexa- orpenta-acylated. The ability of S. typhimurium to palmitate its lipid Aincreases resistance to antimicrobial peptides that are secreted intophagolysozomes.

In wild type S. typhimurium, expression of pagP results in a lipid Athat is hepta-acylated. In an msbB mutant (in which the terminal acylchain of the lipid A cannot be added), the induction of pagP results ina hexa-acylated LPS (Kong et al. (2011) Infection and Immunity79(12):5027-5038). Hexa-acylated LPS has been shown to be the mostpro-inflammatory. While other groups have sought to exploit thispro-inflammatory signal, for example, by deletion of pagP to allow onlyhexa-acylated LPS to be produced (Felgner et al. (2016) Gut Microbes7(2): 171-177; Felgner et al. (2018) Oncoimmunology 7(2): e1382791),this can lead to poor tolerability, due to the TNF-α-mediatedpro-inflammatory nature of the LPS and paradoxically less adaptiveimmunity (Kocijancic et al. (2017) Oncotarget 8(30):49988-50001).

LPS is a potent TLR-4 agonist that induces TNF-α and IL-6. Thedose-limiting toxicities in the I.V. VNP20009 clinical trial (Toso etal. (2002) J. Clin. Oncol. 20(1):142-152) at 1E9 CFU/m² were cytokinemediated (fever hypotension), with TNF-α levels >100,000 pg/ml and IL-6levels >10,000 pg/ml in serum at 2 hr. Despite the msbB deletion inVNP20009 and its reduced pyrogenicity, the LPS still can be toxic athigh doses, possibly due to the presence of hexa-acylated LPS. Thus, apagP⁻/msbB⁻ strain is better tolerated at higher doses, as it cannotproduce hexa-acylated LPS, and will allow for dosing in humans at orabove 1E9 CFU/m². Higher dosing can lead to increased tumorcolonization, enhancing the therapeutic efficacy of theimmunostimulatory bacteria.

Provided herein, are live attenuated Salmonella strains, such as theexemplary strain of S. typhimurium, that only can produce penta-acylatedLPS, that contain a deletion of the msbB gene (that prevents theterminal myristoylation of lipid A, as described above), and thatfurther are modified by deletion of pagP (preventing palmitoylation). Astrain modified to produce penta-acylated LPS allows for lower levels ofpro-inflammatory cytokines, increased sensitivity to antimicrobialpeptides, enhanced tolerability, and increased anti-tumor immunity whenfurther modified to express interfering RNAs against immune checkpointssuch as TREX1.

g. Deletions of SPI-1 and SPI-2 Genes

As described above, pathogenesis, in certain bacterial species,including Salmonella species, such as S. typhimurium, involves a clusterof genes referred to as Salmonella pathogenicity islands (SPIs; see FIG.22) The SPI designated SPI-1 mediates invasion of epithelial cells. Theoperons and genes and their functions are depicted in FIG. 22. SPI-1genes include, but are not limited to: avrA, hilA, hilD, invA, invB,invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ,spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA,sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP.Deletion of one or more of these genes reduces or eliminates the abilityof the bacterium to infect epithelial cells, but does not affect theirability to infect or invade phagocytic cells, including phagocyticimmune cells.

Salmonella invades non-phagocytic intestinal epithelial cells using atype 3 secretion system (T3SS) encoded by the Salmonella pathogenicityisland 1, which forms a needle-like structure that injects effectorproteins directly into the cytosol of host cells. These effectorproteins lead to rearrangement of the eukaryotic cell cytoskeleton tofacilitate invasion of the intestinal epithelium, and also inducesproinflammatory cytokines. The SPI-1 locus includes 39 genes that encodecomponents of this invasion system (see, FIG. 22, reproduced fromKimbrough and Miller (2002) Microbes Infect. 4(1):75-82).

SPI-1 encodes a type 3 secretion system (T3SS) that is responsible fortranslocation of effector proteins into the cytosol of host cells thatcan cause actin rearrangements that lead to uptake of Salmonella. TheSPI-1 T3SS is essential for crossing the gut epithelial layer, but isdispensable for infection when bacteria are injected parenterally. Theinjection of some proteins and the needle complex itself can also induceinflammasome activation and pyroptosis of phagocytic cells. Thispro-inflammatory cell death can limit the initiation of a robustadaptive immune response by directly inducing the death ofantigen-presenting cells (APCs), as well as modifying the cytokinemilieu to prevent the generation of memory T-cells. SPI-1 genes comprisea number of operons including: sitABCD, sprB, avrA, hilC, orgABC,prgKJIH, hilD, hilA, iagB, sptP, sicC, iacP, sipADCB, sicA, spaOPQRS,invFGEABCIJ, and invH.

T3SSs are complexes that play a large role in the infectivity ofGram-negative bacteria, by injecting bacterial protein effectorsdirectly into host cells in an ATP-dependent manner. T3SS complexescross the inner and outer bacterial membranes and create a pore ineukaryotic cell membranes upon contact with a host cell. They consist ofan exportation apparatus, a needle complex and a translocon at the tipof the needle (FIG. 22). The needle complex includes the needle proteinPrgI, a basal body, which anchors the complex in the bacterial membranesand consists of the proteins PrgH, PrgK and InvG, and other proteins,including InvH, PrgJ (rod protein) and InvJ. The translocon, which formsthe pore in the host cell, is a complex of the proteins SipB, SipC andSipD. The exportation apparatus, which allows for the translocation ofthe effector proteins, is comprised of the proteins SpaP, SpaQ, SpaR,SpaS, InvA, InvC and OrgB. A cytoplasmic sorting platform, whichestablishes the specific order of protein secretion, is composed of theproteins SpaO, OrgA and OrgB (Manon et al. (2012), Salmonella, Chapter17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

The effectors translocated into the host cell by T3SS-1 include SipA,SipC, SopB, SopD, SopE, SopE2 and SptP, which are essential for cellinvasion. For example, S. typhimurium sipA mutants exhibit 60-80%decreased invasion, sipC deletion results in a 95% decrease in invasion,and sopB deletion results in a 50% decrease in invasion (Manon et al.(2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp.339-364). Other effectors include AvrA, which controlsSalmonella-induced inflammation. Chaperones, which bind secretedproteins and maintain them in a conformation that is competent forsecretion, include SicA, InvB and SicP. Transcriptional regulatorsinclude HilA, HilD, InvF, SirC and SprB. Unclassified T3SS SPI-1proteins, which have various functions in type III secretion, includeOrgC, InvE, InvI, IacP and IagB (see, FIG. 22, adapted from Kimbrough etal. (2002) Microbes Infect. 4(1):75-82).

Thus, the inactivation of SPI-1-dependent invasion, through theinactivation or knockout of one or more genes involved in the SPI-1pathway, eliminates the ability of the bacteria to infect epithelialcells. These genes include, but are not limited to, one more of: avrA,hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP,iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ,prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2,sprB, and sptP.

Salmonella mutants lacking the T3SS-1 have been shown to invade numerouscell lines/types, by a T3SS-1 independent invasion mechanism, involvingseveral proteins, including the invasins Rck, PagN and HlyE. The rckoperon contains 6 open reading frames: pefI, srgD, srgA, srgB, rck andsrgC. pefI encodes a transcriptional regulator of the pef operon, whichis involved in the biosynthesis of the Pef fimbriae. These fimbriae areinvolved in biofilm formation, adhesion to murine small intestine andfluid accumulation in the infant mouse. SrgA oxidizes the disulfide bondof PefA, the major structural subunit of the Pef fimbriae. srgD encodesa putative transcriptional regulator; SrgD together with PefI work toinduce a synergistic negative regulation of flagellar gene expression.srgB encodes a putative outer membrane protein, and srgC encodes aputative transcriptional regulator (Manon et al. (2012), Salmonella,Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

Rck is a 17 kDa outer membrane protein encoded by the large virulenceplasmid of S. enteritidis and S. typhimurium, that induces adhesion toand invasion of epithelial cells, and confers a high level of resistanceto neutralization by complement, by preventing the formation of themembrane attack complex. An rck mutant exhibited a 2-3 fold decrease inepithelial cell invasion compared to the wild-type strain, while Rckoverexpression leads to increased invasion. Rck induces cell entry by areceptor-mediated process, promoting local actin remodeling and weak andclosely adherent membrane extensions. Thus, Salmonella can enter cellsby two distinct mechanisms: the Trigger mechanism mediated by the T3SS-1complex, and a Zipper mechanism induced by Rck (Manon et al. (2012),Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

The invasin PagN is an outer membrane protein that has also been shownto play a role in Salmonella invasion. pagN expression is regulatedbyphoP. Specific stimuli, for example, acidified macrophage phagosomeenvironments or low Mg²⁺ concentrations, are sensed by PhoQ, which thenactivates PhoP to regulate specific genes. It has been shown that thedeletion of pagN in S. typhimurium results in a 3-fold decrease in theinvasion of enterocytes, without altering cell adhesion. Although thePagN-mediated entry mechanism is not fully understood, it has been shownthat actin polymerization is required for invasion. Studies have shownthat PagN is required for Salmonella survival in BALB/c mice, and that apagN mutant is less competitive for colonizing the spleen of mice thanthe parent strain. Because pagN is activated by PhoP, it is mostlyexpressed intracellularly, where the SPI-1 island encoding T3SS-1 isdownregulated. It is thus possible that bacteria exiting epithelialcells or macrophages have an optimal level of PagN expression, but havelow T3SS-1 expression, which can mediate subsequent interactions withother cells encountered following host cell destruction, indicating arole for PagN in Salmonella pathogenesis (Manon et al. (2012),Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

hlyE shares more than 90% sequence identity with the E. coli HlyE (ClyA)hemolysin. The HlyE protein lyses epithelial cells when exported frombacterial cells via outer membrane vesicle release, and is involved inepithelial cell invasion. HlyE also is involved in the establishment ofsystemic Salmonella infection (Manon et al. (2012), Salmonella, Chapter17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

Elimination of the ability to infect epithelial cells also can beachieved by engineering the immunostimulatory bacteria herein to containknockouts or deletions of genes encoding proteins involved inSPI-1-independent invasion, such as one or more of the genes rck, pagN,hlyE, pefI, srgD, srgA, srgB, and srgC.

As described herein, provided are immunostimulatory bacteria that aremodified so that they do not infect epithelial cells, but retain theability to infect phagocytic cells, including tumor-resident immunecells, thereby effectively targeting the immunostimulatory bacteria tothe tumor microenvironment. This is achieved by deleting or knocking outany of the proteins in SPI-1, including, but are not limited to,deletions of one more of: avrA, hilA, hilD, invA, invB, invC, invE,invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS,orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC,sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP, as well as one ormore of rck, pagN, hlyE, pefI, srgD, srgA, srgB, rck and srgC.

The immunostimulatory bacteria that do not infect epithelial cells canbe further modified as described here to encode products that stimulatethe immune system, including, for example, cytokines. The bacteriagenerally have an asd deletion to render them unable to replicate in amammalian host.

For example, provided are strains of S. typhimurium modified by deletionof one or more SPI-1 genes, and also modified by one or more of a purIdeletion, an msbB deletion, and an asd deletion, and by deliveringplasmids encoding proteins that stimulate the immune system, such asgenes encoding immunostimulatory cytokines. For example, bacteria withdeletions of a regulatory gene (e.g., hilA or invF) required forexpression of the SPI-1-associated type 3 secretion system (T3SS-1), aT3SS-1 structural gene (e.g., invG or prgH), and/or a T3SS-1 effectorgene (e.g., sipA or avrA) are provided. As discussed above, thissecretion system is responsible for injecting effector proteins into thecytosol of non-phagocytic host cells such as epithelial cells that causethe uptake of the bacteria; deletion of one or more of these geneseliminates infection/invasion of epithelial cells. Deletion of one ormore of the genes, such as hilA, provides immunostimulatory bacteriathat can be administered intravenously or intratumorally, resulting ininfection of phagocytic cells, which do not require the SPI-1 T3SS foruptake, and also prolongs the longevity of these phagocytic cells. ThehilA mutation also reduces the quantity of pro-inflammatory cytokines,increasing the tolerability of the therapy, as well as the quality ofthe adaptive immune response.

Salmonella also have a Salmonella pathogenicity island 2 (SPI-2),encoding another T3SS that is activated following entry of the bacteriuminto the host cell, and interferes with phagosome maturation, resultingin the formation of a specialized Salmonella-containing vacuole (SCV),where the Salmonella resides during intracellular survival andreplication. SPI-2 T3SS effectors include SseB, SseC, SseD and SpiC,which are responsible for assembly of the F-actin coat aroundintracellular bacteria; this actin coat promotes fusion of the SCV withactin-containing or actin-propelled vesicles, and prevents it fromfusing with unfavorable compartments. SifA is responsible for theformation of Salmonella-induced filaments (SIFs), which are tubules thatconnect the individual SCVs in the infected cell. sifA is essential tomaintaining the integrity of the SCV, and sifA mutants are released intothe cytosol of host cells. SseF and SseG are components of the SPI-2T3SS that are involved in SCV positioning and cellular traffickingprocesses that direct materials required for the bacterium's survivaland replication to the SCV. SseF and SseG also are involved in SIFformation. Other SPI-2 T3SS effectors include PipB2, SopD2, and SseJ,which are involved in SIF and SCV formation, and maintenance of vacuoleintegrity; SpvC, SseL, and SspH1, which are involved in host immunesignaling; and SteC, SspH2, SrfH/Ssel and SpvB, which are involved inthe formation of the SCV F-actin meshwork, in the migration of infectedphagocytes, in the inhibition of actin polymerization, and in P-bodydisassembly in infected cells (Coburn et al. (2007) ClinicalMicrobiology Reviews 20(4):535-549; Figueira and Holden (2012)Microbiology 158:1147-1161).

The immunostimulatory bacteria herein can include deletions ormodifications in any of the SPI-2 T3SS genes that affect the formationor integrity of the SCV and associated structures, such as SIFs. Thesemutants have an increased frequency of SCV escape and can replicate inthe cytosol. For example, immunostimulatory bacteria, such as Salmonellaspecies, engineered to escape the SCV are more efficient at deliveringmacromolecules, such as plasmids, as the lipid bilayer of the SCV is apotential barrier. This is achieved by deletion or mutation of genesrequired for Salmonella induced filament (SIF) formation, including, forexample, sifA, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA

The immunostimulatory bacteria that can escape the SCV can be furthermodified as described here to encode products that stimulate the immunesystem, including, for example, cytokines. The bacteria generally havean asd deletion to render them unable to replicate in a mammalian host.

h. Endonuclease (endA) Mutations to Increase Plasmid Delivery

The endA gene (for example, SEQ ID NO:250) encodes an endonuclease (forexample, SEQ ID NO:251) that mediates degradation of double stranded DNAin the periplasm of Gram negative bacteria. Most common strains oflaboratory E. coli are endA−, as a mutation in the endA gene allows forhigher yields of plasmid DNA. This gene is conserved among species. Tofacilitate intact plasmid DNA delivery, the endA gene of the engineeredimmunostimulatory bacteria is deleted or mutated to prevent itsendonuclease activity. Exemplary of such mutations is an E208K aminoacid substitution (Durfee, et al. (2008) J Bacteriol. 190(7):2597-2606)or a corresponding mutation in the species of interest. endA, includingE208, is conserved among bacterial species, including Salmonella. Thus,the E208K mutation can be used to eliminate endonuclease activity inother species, including Salmonella species. Those of skill in the artcan introduce other mutations or deletions to eliminate endA activity.Effecting this mutation or deleting or disrupting the gene to eliminateactivity of the endA in the immunostimulatory bacteria herein, such asin Salmonella, increases efficiency of intact plasmid DNA delivery,thereby increasing expression of the RNAs, such as the shRNA and/ormiRNA, targeting any or two or more of the immune checkpoints, encodedin the plasmid, thereby increasing RNAi-mediated knockdown of checkpointgenes and enhancing anti-tumor efficacy.

i. RIG-I Inhibition

Of the TLR-independent type I IFN pathways, one is mediated by hostrecognition of single-stranded (ss) and double-stranded (ds) RNA in thecytosol. These are sensed by RNA helicases, including retinoicacid-inducible gene I (RIG-I), melanoma differentiation-associated gene5 (MDA-5), and through the IFN-β promoter stimulator 1 (IPS-1) adaptorprotein-mediated phosphorylation of the IRF-3 transcription factor,leading to induction of type I IFN (Ireton and Gale (2011) Viruses3(6):906-919). RIG-I recognizes dsRNA and ssRNA bearing5′-triphosphates. This moiety can directly bind RIG-I, or be synthesizedfrom a poly(dA-dT) template by the poly DNA-dependent RNA polymerase III(Pol III) (Chiu, Y. H. et al. (2009) Cell 138(3):576-91). A poly(dA-dT)template containing two AA dinucleotide sequences occurs at the U6promoter transcription start site in a common lentiviral shRNA cloningvector. Its subsequent deletion in the plasmid prevents type I IFNactivation (Pebernard et al. (2004) Differentiation. 72:103-111). ARIG-I binding sequence can be included in the plasmids provided herein;inclusion can increase immunostimulation that increases anti-tumoralactivity of the immunostimulatory bacteria herein.

j. DNase II Inhibition

Another nuclease responsible for degrading foreign and self DNA is DNaseII, an endonuclease, which resides in the endosomal compartment anddegrades DNA following apoptosis. Lack of DNase II (Dnase2a in mice)results in the accumulation of endosomal DNA that escapes to the cytosoland activates cGAS/STING signaling (Lan Y Y et al. (2014) Cell Rep.9(1):180-192). Similar to TREX1, DNase II-deficiency in humans presentswith autoimmune type I interferonopathies. In cancer, dying tumor cellsthat are engulfed by tumor-resident macrophages prevent cGAS/STINGactivation and potential autoimmunity through DNase II digestion of DNAwithin the endosomal compartment (Ahn et al. (2018) Cancer Cell33:862-873). Hence, embodiments of the immunostimulatory bacterialstrains, as provided herein, encode RNAi, such as shRNA or miRNA thatinhibit, suppress or disrupt expression of DNase II, which can inhibitDNase II in the tumor microenvironment, thereby provoking accumulationof endocytosed apoptotic tumor DNA in the cytosol, where it can act as apotent cGAS/STING agonist

k. RNase H2 Inhibition

While TREX1 and DNase II function to clear aberrant DNA accumulation,RNase H2 functions similarly to eliminate pathogenic accumulation ofRNA:DNA hybrids in the cytosol. Similar to TREX1, deficiencies in RNaseH2 also contribute to the autoimmune phenotype of Aicardi-Goutieressyndrome (Rabe, B. (2013) J. Mol. Med. 91:1235-1240). Specifically, lossof RNase H2 and subsequent accumulation of RNA:DNA hybrids orgenome-embedded ribonucleotide substrates has been shown to activatecGAS/STING signaling. (MacKenzie et al. (2016) EMBO J. April 15;35(8):831-44). Hence, embodiments of the immunostimulatory bacterialstrains, as provided herein, encode RNAi, such as shRNA or miRNA thatinhibit, suppress or disrupt expression of RNase H2, to thereby inhibitRNase H2, resulting in tumor-derived RNA:DNA hybrids and derivativesthereof, which activate cGAS/STING signaling and anti-tumor immunity.

l. Stabilin-1/CLEVER-1 Inhibition

Another molecule expressed primarily on monocytes and involved inregulating immunity is stabilin-1 (gene name STAB1, also known asCLEVER-1, FEEL-1). Stabilin-1 is a type I transmembrane protein that isupregulated on endothelial cells and macrophages following inflammation,and in particular, on tumor-associated macrophages (Kzhyshkowska et al.(2006) J. Cell. Mol. Med. 10(3):635-649). Upon inflammatory activation,stabilin-1 acts as a scavenger and aids in wound healing and apoptoticbody clearance, and can prevent tissue injury, such as liver fibrosis(Rantakari et al. (2016) Proc. Natl. Acad. Sci. USA 113(33):9298-9303).Upregulation of stabilin-1 directly inhibits antigen-specific T cellresponses, and knockdown by siRNA in monocytes was shown to enhancetheir pro-inflammatory function (Palani, S. et al. (2016) J. Immunol.196:115-123). Hence, embodiments of the immunostimulatory bacterialstrains, as provided herein, encode RNAi, such as shRNA or miRNA thatinhibit, suppress or disrupt expression of Stabilin-1/CLEVER-1 in thetumor microenvironment, thereby enhancing the pro-inflammatory functionsof tumor-resident macrophages.

5. Immunostimulatory Proteins

The immunostimulatory bacteria herein can be modified to encode animmunostimulatory protein that promotes or induces or enhances ananti-tumor response. The immunostimulatory protein can be encoded on aplasmid in the bacterium, under the control of a eukaryotic promoter,such as a promoter recognized by RNA polymerase II, for expression in aeukaryotic subject, particularly the subject for whom theimmunostimulatory bacterium is to be administered, such as a human. Thenucleic acid encoding the immunostimulatory protein can include, inaddition to the eukaryotic promoter, other regulatory signals forexpression or trafficking in the cells, such as for secretion orexpression on the surface of a cell.

Immunostimulatory proteins are those that, in the appropriateenvironment, such as a tumor microenvironment (TME), can promote orparticipate in or enhance an anti-tumor response by the subject to whomthe immunostimulatory bacterium is administered. Immunostimulatoryproteins include, but are not limited to, cytokines, chemokines andco-stimulatory molecules. These include cytokines, such as, but notlimited to, IL-2, IL-7, IL-12, IL-15, and IL-18; chemokines, such as,but not limited to, CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11; and/orco-stimulatory molecules, such as, but not limited to, CD40, CD40L,OX40, OX40L, 4-1BB, 4-1BBL, members of the TNF/TNFR superfamily andmembers of the B7-CD28 family. Other such immunostimulatory proteinsthat are used for treatment of tumors or that can promote, enhance orotherwise increase or evoke an anti-tumor response, known to those ofskill in the art, are contemplated for encoding in the immunostimulatorybacteria provided herein.

The genome of the immunostimulatory bacteria provided herein also can bemodified to increase or promote infection of immune cells, particularlyimmune cells in the tumor microenvironment, such as phagocytic cells.The bacteria also can be modified to decrease pyroptosis in immunecells. The immunostimulatory bacteria include those, for example, thathave modifications that disrupt/inhibit the SPI-1 pathway, such asdisruption or deletion of hilA, and/or disruption/deletion of flagellingenes, rod protein, needle protein, and/or pagP as detailed andexemplified elsewhere herein.

Immunostimulatory Bacteria Encoding Cytokines and Chemokines

In some embodiments, the immunostimulatory bacteria herein areengineered to express cytokines to stimulate the immune system,including, but not limited to, IL-2, IL-7, IL-12 (IL-12p70 (IL12p40+IL-12p35)), IL-15 (and the IL-15:IL-15R alpha chain complex), andIL-18. Cytokines stimulate immune effector cells and stromal cells atthe tumor site, and enhance tumor cell recognition by cytotoxic cells.In some embodiments, the immunostimulatory bacteria can be engineered toexpress chemokines, such as, for example, CCL3, CCL4, CCL5, CXCL9,CXCL10 and CXCL11.

IL-2

Interleukin-2 (IL-2), which was the first cytokine approved for thetreatment of cancer, is implicated in the activation of the immunesystem by several mechanisms, including the activation and promotion ofCTL growth, the generation of lymphokine-activated killer (LAK) cells,the promotion of Treg cell growth and proliferation, the stimulation ofTILs, and the promotion of T cell, B cell and NK cell proliferation anddifferentiation. Recombinant IL-2 (rIL-2) is FDA-approved for thetreatment of metastatic renal cell carcinoma (RCC) and metastaticmelanoma (Sheikhi et al. (2016) Iran J. Immunol. 13(3):148-166).

IL-7

IL-7, which is a member of the IL-2 superfamily, is implicated in thesurvival, proliferation and homeostasis of T cells. Mutations in theIL-7 receptor have been shown to result in the loss of T cells, and thedevelopment of severe combined immunodeficiency (SCID), highlighting thecritical role that IL-7 plays in T cell development. IL-7 is ahomeostatic cytokine that provides continuous signals to resting naïveand memory T cells, and which accumulates during conditions oflymphopenia, leading to an increase in both T cell proliferation and Tcell repertoire diversity. In comparison to IL-2, IL-7 is selective forexpanding CD8⁺ T cells over CD4⁺FOXP3⁺ regulatory T cells. RecombinantIL-7 has been shown to augment antigen-specific T cell responsesfollowing vaccination and adoptive cell therapy in mice. IL-7 also canplay a role in promoting T-cell recovery following chemotherapy ofhematopoietic stem cell transplantation. Early phase clinical trials onpatients with advanced malignancy have shown that recombinant IL-7 iswell-tolerated and has limited toxicity at biologically active doses(i.e., in which the numbers of circulating CD4⁺ and CD8⁺ T cellsincreased by 3-4 fold) (Lee, S. and Margolin, K. (2011) Cancers3:3856-3893). IL-7 has been shown to possess antitumor effects in tumorssuch as gliomas, melanomas, lymphomas, leukemia, prostate cancer andglioblastoma, and the in vivo administration of IL-7 in murine modelsresulted in decreased cancer cell growth. IL-7 also has been shown toenhance the antitumor effects of IFN-γ in rat glioma tumors, and toinduce the production of IL-1α, IL-1β and TNF-α by monocytes, whichresults in the inhibition of melanoma growth. Additionally,administration of recombinant IL-7 following the treatment of pediatricsarcomas resulted in the promotion of immune recovery (Lin et al. (2017)Anticancer Research 37:963-968).

IL-12 (IL-12p70 (IL-12p40+IL-12p35)

Bioactive IL-12 (IL-12p70), which promotes cell-mediated immunity, is aheterodimer, composed of p35 and p40 subunits, whereas IL-12p40 monomersand homodimers act as IL-12 antagonists. IL-12, which is secreted byantigen-presenting cells, promotes the secretion of IFN-γ from NK and Tcells, inhibits tumor angiogenesis, results in the activation andproliferation of NK cells, CD8⁺ T cells and CD4⁺ T cells, enhances thedifferentiation of CD4⁺ Th0 cells into Th1 cells, and promotesantibody-dependent cell-mediated cytotoxicity (ADCC) against tumorcells. IL-12 has been shown to exhibit antitumor effects in murinemodels of melanoma, colon carcinoma, mammary carcinoma and sarcoma(Kalinski et al. (2001) Blood 97:3466-3469; Sheikhi et al. (2016) IranJ. Immunol. 13(3):148-166; Lee, S. and Margolin, K. (2011) Cancers3:3856-3893).

IL-15 and IL-15:IL-15Rα

IL-15 is structurally similar to IL-2, and while both IL-2 and IL-15provide early stimulation for the proliferation and activation of Tcells, IL-15 blocks IL-2 induced apoptosis, which is a process thatleads to the elimination of stimulated T cells and induction of T-celltolerance, limiting memory T cell responses and potentially limiting thetherapeutic efficacy of IL-2 alone. IL-15 also supports the persistenceof memory CD8⁺ T cells for maintaining long-term antitumor immunity, andhas demonstrated significant antitumor activity in pre-clinical murinemodels via the direct activation of CD8⁺ effector T cells in anantigen-independent manner. In addition to CD8⁺ T cells, IL-15 isresponsible for the development, proliferation and activation ofeffector natural killer (NK) cells (Lee, S. and Margolin, K. (2011)Cancers 3:3856-3893; Han et al. (2011) Cytokine 56(3):804-810).

IL-15 and IL-15 receptor alpha (IL-15Rα) are coordinately expressed byantigen-presenting cells such as monocytes and dendritic cells, andIL-15 is presented in trans by IL-15Rα to the IL-15Rβγ_(C) receptorcomplex expressed on the surfaces of CD8⁺ T cells and NK cells. SolubleIL-15:IL15-Rα complexes have been shown to modulate immune responses viathe IL-15Rβγ_(C) complex, and the biological activity of IL-15 has beenshown to be increased 50-fold by administering it in a preformed complexof IL-15 and soluble IL-15Rα, which has an increased half-life comparedto IL-15 alone. This significant increase in the therapeutic efficacy ofIL-15 by pre-association with IL-15Rα has been demonstrated in murinetumor models (Han et al. (2011) Cytokine 56(3):804-810).

IL-18

IL-18 induces the secretion of IFN-γ by NK and CD8⁺ T cells, enhancingtheir toxicity. IL-18 also activates macrophages and stimulates thedevelopment of Th1 helper CD4⁺ T cells. IL-18 has shown promisinganti-tumor activity in several preclinical mouse models. For example,administration of recombinant IL-18 (rIL-18) resulted in the regressionof melanoma or sarcoma in syngeneic mice through the activation of CD4⁺T cells and/or NK cell-mediated responses. Other studies showed thatIL-18 anti-tumor effects were mediated by IFN-γ and involvedantiangiogenic mechanisms. The combination of IL-18 with othercytokines, such as IL-12, or with co-stimulatory molecules, such asCD80, enhances the IL-18-mediated anti-tumor effects. Phase I clinicaltrials in patients with advanced solid tumors and lymphomas showed thatIL-18 administration was safe, and that it resulted in immune modulatoryactivity and in the increase of serum IFN-γ and GM-CSF levels inpatients and modest clinical responses. Clinical trials showed thatIL-18 can be combined with other anticancer therapeutic agents, such asmonoclonal antibodies, cytotoxic drugs or vaccines (Fabbi et al. (2015)J. Leukoc. Biol. 97:665-675; Lee, S. and Margolin, K. (2011) Cancers3:3856-3893).

It was found that an attenuated strain of Salmonella typhimurium,engineered to express IL-18, inhibited the growth of s.c. tumors orpulmonary metastases in syngeneic mice without any toxic effectsfollowing systemic administration. Treatment with this engineeredbacterium induced the accumulation of T cells, NK cells and granulocytesin tumors, and resulted in the intratumoral production of cytokines(Fabbi et al. (2015) J. Leukoc. Biol. 97:665-675).

Chemokines

Chemokines are a family of small cytokines that mediate leukocytemigration to areas of injury or inflammation and are involved inmediating immune and inflammatory responses. Chemokines are classifiedinto four subfamilies, based on the position of cysteine residues intheir sequences, namely XC-, CC-, CXC- and CX3C-chemokine ligands, orXCL, CCL, CXCL and CX3CL. The chemokine ligands bind to their cognatereceptors and regulate the circulation, homing and retention of immunecells, with each chemokine ligand-receptor pair selectively regulating acertain type of immune cell. Different chemokines attract differentleukocyte populations, and form a concentration gradient in vivo, withattracted immune cells moving through the gradient towards the higherconcentration of chemokine (Argyle D. and Kitamura, T. (2018) Front.Immunol. 9:2629; Dubinett et al. (2010) Cancer J. 16(4):325-335).Chemokines can improve the antitumor immune response by increasing theinfiltration of immune cells into the tumor, and facilitating themovement of antigen-presenting cells (APCs) to tumor-draining lymphnodes, which primes naïve T cells and B cells (Lechner et al. (2011)Immunotherapy 3(11): 1317-1340). The immunostimulatory bacteria hereincan be engineered to encode chemokines, including, but not limited to,CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11.

CCL3, CCL4, CCL5

CCL3, CCL4 and CCL5 share a high degree of homology, and bind to CCR5(CCL3, CCL4 and CCL5) and CCR1 (CCL3 and CCL5) on several cell types,including immature DCs and T cells, in both humans and mice. TherapeuticT cells have been shown to induce chemotaxis of innate immune cells totumor sites, via the tumor-specific secretion of CCL3, CCL4 and CCL5(Dubinett et al. (2010) Cancer J. 16(4):325-335).

The induction of the T helper cell type 1 (Th1) response releases CCL3.In vivo and in vitro studies of mice have indicated that CCL3 ischemotactic for both neutrophils and monocytes; specifically, CCL3 canmediate myeloid precursor cell (MPC) mobilization from the bone marrow,and has MPC regulatory and stimulatory effects. Human ovarian carcinomacells transfected with CCL3 showed enhanced T cell infiltration andmacrophages within the tumor, leading to an improved antitumor response,and indicated that CCL3-mediated chemotaxis of neutrophils suppressedtumor growth. DCs transfected with the tumor antigen humanmelanoma-associated gene (MAGE)-1 that were recruited by CCL3 exhibitedsuperior anti-tumor effects, including increased lymphocyteproliferation, cytolytic capacity, survival, and decreased tumor growthin a mouse model of melanoma. A combinatorial use of CCL3 with anantigen-specific platform for MAGE-1 has also been used in the treatmentof gastric cancer. CCL3 production by CT26, a highly immunogenic murinecolon tumor, slowed in vivo tumor growth; this process was indicated tobe driven by the CCL3-dependent accumulation of natural killer (NK)cells, and thus, IFNγ, resulting in the production of CXCL9 and CXLC 10(Allen et al. (2017) Oncoimmunology 7(3):e1393598; Schaller et al.(2017) Expert Rev. Clin. Immunol. 13(11):1049-1060).

CCL3 has been used as an adjuvant for the treatment of cancer.Administration of a CCL3 active variant, ECI301, after radiofrequencyablation in mouse hepatocellular carcinoma increased tumor-specificresponses, and this mechanism was further shown to be dependent on theexpression of CCR1. CCL3 has also shown success as an adjuvant insystemic cancers, whereby mice vaccinated with CCL3 and IL-2 orgranulocyte-macrophage colony-stimulating factor (GM-CSF) in a model ofleukemia/lymphoma exhibited increased survival (Schaller et al. (2017)Expert Rev. Clin. Immunol. 13(11): 1049-1060).

CCL3 and CCL4 play a role in directing CD8⁺ T cell infiltration intoprimary tumor sites in melanoma and colon cancers. Tumor production ofCCL4 leads to the accumulation of CD103⁺ DCs; suppression of CCL4through a WNT/β-catenin-dependent pathway prevented CD103⁺ DCinfiltration of melanoma tumors (Spranger et al. (2015) Nature523(7559):231-235). CCL3 was also shown to enhance CD4⁺ and CD8⁺ T cellinfiltration to the primary tumor site in a mouse model of colon cancer(Allen et al. (2017) Oncoimmunology 7(3):e1393598).

The binding of CCL3 or CCL5 to their receptors (CCR1 and CCR5,respectively), moves immature DCs, monocytes and memory and T effectorcells from the circulation into sites of inflammation or infection. Forexample, CCL5 expression in colorectal tumors contributes to Tlymphocyte chemoattraction and survival. CCL3 and CCL5 have been usedalone or in combination therapy to induce tumor regression and immunityin several preclinical models. For example, studies have shown that thesubcutaneous injection of Chinese hamster ovary cells geneticallymodified to express CCL3 resulted in tumor inhibition and neutrophilicinfiltration. In another study, a recombinant oncolytic adenovirusexpression CCL5 (Ad-RANTES-E1A) resulted in primary tumor regression andblocked metastasis in a mammary carcinoma murine model (Lechner et al.(2011) Immunotherapy 3(11): 1317-1340).

In a translational study of colorectal cancer, CCL5 induced an“antiviral response pattern” in macrophages. As a result of CXCR3mediated migration of lymphocytes at the invasive margin of livermetastases in colorectal cancer, CCL5 is produced. Blockade of CCR5, theCCL5 receptor, results in tumor death, driven by macrophages producingIFN and reactive oxygen species. While macrophages are present in thetumor microenvironment, CCR5 inhibition induces a phenotypic shift froman M2 to an M1 phenotype. CCR5 blockade also leads to clinical responsesin colorectal cancer patients (Halama et al. (2016) Cancer Cell29(4):587-601).

CCL3, CCL4 and CCL5 can be used treating conditions including lymphatictumors, bladder cancer, colorectal cancer, lung cancer, melanoma,pancreatic cancer, ovarian cancer, cervical cancer or liver cancer (U.S.Patent Publication No. US 2015/0232880; International Patent PublicationNos. WO 2015/059303, WO 2017/043815, WO 2017/156349 and WO 2018/191654).

CXCL9, CXCL10, CXCL11

CXCL9 (MIG), CXCL10 (IP10) and CXCL11 (ITAC) are induced by theproduction of IFN-γ. These chemokines bind CXCR3, preferentiallyexpressed on activated T cells, and function both angiostatically and inthe recruitment and activation of leukocytes. Prognosis in colorectalcancer is strongly correlated to tumor-infiltrating T cells,particularly Th1 and CD8⁺ effector T cells; high intratumoral expressionof CXCL9, CXCL10 and CXCL11 is indicative of good prognosis. Forexample, in a sample of 163 patients with colon cancer, those with highlevels of CXCL9 or CXCL11 showed increased post-operative survival, andpatients with high CXC expression had significantly higher numbers ofCD3⁺ T-cells, CD4⁺ T-helper cells, and CD8⁺ cytotoxic T-cells. In livermetastases of colorectal cancer patients, CXCL9 and CXCL10 levels wereincreased at the invasive margin and correlated with effector T celldensity. The stimulation of lymphocyte migration via the action of CXCL9and CXCL10 on CXCR3 leads to the production of CCL5 at the invasivemargin (Halama et al. (2016) Cancer Cell 29(4):587-601; Kistner et al.(2017) Oncotarget 8(52):89998-90012).

In vivo, CXCL9 functions as a chemoattractant for tumor-infiltratinglymphocytes, activated peripheral blood lymphocytes, natural killer (NK)cells and Th1 lymphocytes. CXCL9 also is critical for T cell-mediatedsuppression of cutaneous tumors. For example, when combined withsystemic IL-2, CXCL9 has been shown to inhibit tumor growth via theincreased intratumoral infiltration of CXCR3⁺ mononuclear cells. In amurine model of colon carcinoma, a combination of the huKS1/4-IL-2fusion protein with CXCL9 gene therapy achieved a superior anti-tumoreffect and prolonged lifespan through the chemoattraction and activationof CD8⁺ and CD4⁺ T lymphocytes (Dubinett et al. (2010) Cancer J.16(4):325-335; Ruelmann et al. (2001) Cancer Res. 61(23):8498-8503).

CXCL10, produced by activated monocytes, fibroblasts, endothelial cellsand keratinocytes, is chemotactic for activated T cells and can act asan inhibitor of angiogenesis in vivo. Expression of CXCL10 in colorectaltumors has been shown to contribute to cytotoxic T lymphocytechemoattraction and longer survival. The administration ofimmunostimulatory cytokines, such as IL-12, has been shown to enhancethe antitumor effects generated by CXCL10. A DC vaccine primed with atumor cell lysate and transfected with CXCL10 had increasedimmunological protection and effectiveness in mice; the animals showed aresistance to a tumor challenge, a slowing of tumor growth and longersurvival time. In vivo and in vitro studies in mice using theCXCL10-mucin-GPI fusion protein resulted in tumors with higher levels ofrecruited NK cells compared to tumors not treated with the fusionprotein. Interferons (which can be produced by plasmacytoid dendriticcells; these cells are associated with primary melanoma lesions and canbe recruited to a tumor site by CCL20) can act on tumor DC subsets, forexample, CD103⁺ DCs, which have been shown to produce CXCL9/10 in amouse melanoma model and were associated with CXCL9/10 in human disease.CXCL10 also has shown higher expression in human metastatic melanomasamples relative to primary melanoma samples. Therapeutically, adjuvantIFN-α melanoma therapy upregulates CXCL10 production, whereas thechemotherapy agent cisplatin induces CXCL9 and CXCL10 (Dubinett et al.(2010) Cancer J. 16(4):325-335; Kuo et al. (2018) Front. Med. (Lausanne)5:271; Li et al. (2007) Scand. J. Immunol. 65(1):8-13; Muenchmeier etal. (2013) PLoS One 8(8):e72749).

CXCL10/11 and CXCR3 expression has been established in humankeratinocytes derived from basal cell carcinomas (BCCs). CXCL11 also iscapable of promoting immunosuppressive indoleamine 2,3-dioxygenase (IDO)expression in human basal cell carcinoma as well as enhancingkeratinocyte proliferation, which could reduce the anti-tumor activityof any infiltrating CXCR3⁺ effector T cells (Kuo et al. (2018) Front.Med. (Lausanne) 5:271).

CXCL9, CXCL10 and CXCL11 can be encoded in oncolytic viruses fortreating cancer (U.S. Patent Publication No. US 2015/0232880;International Patent Publication No. WO 2015/059303). Pseudotypedoncolytic viruses or a genetically engineered bacterium encoding thegene for CXCL10 also can be used to treat cancer (International PatentPublication Nos. WO 2018/006005 and WO 2018/129404).

Co-Stimulatory Molecules

Co-stimulatory molecules enhance the immune response against tumorcells, and co-stimulatory pathways are inhibited by tumor cells topromote tumorigenesis. The immunostimulatory bacteria herein can beengineered to express co-stimulatory molecules, such as, for example,CD40, CD40L, 4-1BB, 4-1BBL, OX40 (CD134), OX40L (CD252), other membersof the TNFR superfamily (e.g., CD27, GITR, CD30, Fas receptor, TRAIL-R,TNF-R, HVEM, RANK), B7 and CD28. The immunostimulatory bacteria hereinalso can be engineered to express agonistic antibodies againstco-stimulatory molecules to enhance the anti-tumor immune response.

TNF Receptor Superfamily

The TNF superfamily of ligands (TNFSF) and their receptors (TNFRSF) areinvolved in the proliferation, differentiation, activation and survivalof tumor and immune effector cells. Members of this family include CD30,Fas-L, TRAIL-R and TNF-R, which induce apoptosis, and CD27, OX40L,CD40L, GITR-L and 4-1BBL, which regulate B and T cell immune responses.Other members include herpesvirus entry mediator (HVEM) and CD27. Theexpression of TNFSF and TNFRSF by the immunostimulatory bacteria hereincan enhance the antitumor immune response. It has been shown, forexample, that the expression of 4-1BBL in murine tumors enhancesimmunogenicity, and intratumoral injection of dendritic cells (DCs) withincreased expression of OX40L can result in tumor rejection in murinemodels. Studies have also shown that injection of an adenovirusexpressing recombinant GITR into B16 melanoma cells promotes T cellinfiltration and reduces tumor volume. Stimulatory antibodies againstmolecules such as 4-1BB, OX40 and GITR also can be encoded by theimmunostimulatory bacteria to stimulate the immune system. For example,agonistic anti-4-1BB monoclonal antibodies have been shown to enhanceanti-tumor CTL responses, and agonistic anti-OX40 antibodies have beenshown to increase anti-tumor activity in transplantable tumor models.Additionally, agonistic anti-GITR antibodies have been shown to enhanceanti-tumor responses and immunity (Lechner et al. (2011) Immunotherapy3(11):1317-1340; Peggs et al. (2009) Clinical and ExperimentalImmunology 157:9-19).

CD40 and CD40L

CD40, which is a member of the TNF receptor superfamily, is expressed byAPCs and B cells, while its ligand, CD40L (CD 154), is expressed byactivated T cells. Interaction between CD40 and CD40L stimulates B cellsto produce cytokines, resulting in T cell activation and tumor celldeath. Studies have shown that antitumor immune responses are impairedwith reduced expression of CD40L on T cells or CD40 on dendritic cells.CD40 is expressed on the surface of several B-cell tumors, such asfollicular lymphoma, Burkitt lymphoma, lymphoblastic leukemia, andchronic lymphocytic leukemia, and its interaction with CD40L has beenshown to increase the expression of B7.1/CD80, B7.2/CD86 and HLA classII molecules in the CD40⁺ tumor cells, as well as enhance theirantigen-presenting abilities. Transgenic expression of CD40L in a murinemodel of multiple myeloma resulted in the induction of CD4⁺ and CD8⁺ Tcells, local and systemic antitumor immune responses and reduced tumorgrowth. Anti-CD40 agonistic antibodies also induced anti-tumor T cellresponses (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology11:39; Dotti et al. (2002) Blood 100(1):200-207; Murugaiyan et al.(2007) J. Immunol. 178:2047-2055).

4-1BB and 4-1BBL

4-1BB (CD137) is an inducible co-stimulatory receptor that is expressedby T cells, NK cells and APCs, including DCs, B cells and monocytes,which binds its ligand, 4-1BBL to trigger immune cell proliferation andactivation. 4-1BB results in longer and more wide spread responses ofactivated T cells. Anti-4-1BB agonists and 4-1BBL fusion proteins havebeen shown to increase immune-mediated antitumor activity, for example,against sarcoma and mastocytoma tumors, mediated by CD4⁺ and CD⁺ T cellsand tumor-specific CTL activity (Lechner et al. (2011) Immunotherapy3(11): 1317-1340; Marin-Acevedo et al. (2018) Journal of Hematology &Oncology 11:39).

OX40 and OX40L

OX40 (CD 134) is a member of the TNF receptor superfamily that isexpressed on activated effector T cells, while its ligand, OX40L isexpressed on APCs, including DCs, B cells and macrophages, followingactivation by TLR agonists and CD40-CD40L signaling. OX40-OX40Lsignaling results in the activation, potentiation, proliferation andsurvival of T cells, as well as the modulation of NK cell function andinhibition of the suppressive activity of Tregs. Signaling through OX40also results in the secretion of cytokines (IL-2, IL-4, IL-5 and IFN-γ),boosting Th1 and Th2 cell responses. The recognition of tumor antigensby TILs results in increased expression of OX40 by the TILs, which hasbeen correlated with improved prognosis. Studies have demonstrated thattreatment with anti-OX40 agonist antibodies or Fc-OX40L fusion proteinsresults in enhanced tumor-specific CD4⁺ T cell responses and increasedsurvival in murine models of melanoma, sarcoma, colon carcinoma andbreast cancer, while Fc-OX40L incorporated into tumor cell vaccinesprotected mice from subsequent challenge with breast carcinoma cells(Lechner et al. (2011) Immunotherapy 3(11): 1317-1340; Marin-Acevedo etal. (2018) Journal of Hematology & Oncology 11:39).

B7-CD28 Family

CD28 is a costimulatory molecule expressed on the surface of T cellsthat acts as a receptor for B7-1 (CD80) and B7-2 (CD86), which areco-stimulatory molecules expressed on antigen-presenting cells. CD28-B7signaling is required for T cell activation and survival, and preventionof T cell anergy, and results in the production of interleukins such asIL-6.

Optimal T-cell priming requires two signals: (1) T-cell receptor (TCR)recognition of MHC-presented antigens and (2) co-stimulatory signalsresulting from the ligation of T-cell CD28 with B7-1 (CD80) or B7-2(CD86) expressed on APCs. Following T cell activation, CTLA-4 receptorsare induced, which then outcompete CD28 for binding to B7-1 and B7-2ligands. Antigen presentation by tumor cells is poor due to their lackof expression of costimulatory molecules such as B7-1/CD80 andB7-2/CD86, resulting in a failure to activate the T-cell receptorcomplex. As a result, upregulation of these molecules on the surfaces oftumor cells can enhance their immunogenicity. Immunotherapy of solidtumors and hematologic malignancies has been successfully induced by B7,for example, via tumor cell expression of B7, or solubleB7-immunoglobulin fusion proteins. The viral-mediated tumor expressionof B7, in combination with other co-stimulatory ligands such as ICAM-3and LFA-3, has been successful in preclinical and clinical trials forthe treatment of chronic lymphocytic leukemia and metastatic melanoma.Additionally, soluble B7 fusion proteins have demonstrated promisingresults in the immunotherapy of solid tumors as single agentimmunotherapies (Lechner et al. (2011) Immunotherapy 3(11): 1317-1340;Dotti et al. (2002) Blood 100(1):200-207).

6. Modifications that Increase Uptake of Gram-Negative Bacteria, Such asSalmonella, by Immune Cells and Reduce Immune Cell Death

The genome of the immunostimulatory bacteria provided herein can bemodified to increase or promote infection of immune cells, particularlyimmune cells in the tumor microenvironment, such as phagocytic cells.This includes reducing infection of non-immune cells, such as epithelialcells, or increasing infection of immune cells. The bacteria also can bemodified to decrease pyroptosis in immune cells. Numerous modificationsof the bacterial genome can do one or both of increasing infection ofimmune cells and decreasing pyroptosis. The immunostimulatory bacteriaprovided herein include such modifications, for example, deletionsand/or disruptions of genes involved in the SPI-1 T3SS pathway, such asdisruption or deletion of hilA, and/or disruption/deletion of genesencoding flagellin, rod protein and needle protein.

The invasive phenotype of Gram-negative bacteria, such as Salmonella,can result from the activity of genes encoded in pathways that promotethe invasion of host cells. The invasion-associated Salmonellapathogenicity island-1 (SPI-1) of Salmonella is exemplary. SPI-1includes the type 3 secretion system (T3SS), that is responsible fortranslocation of effector proteins into the cytosol of host cells. Theseproteins can cause actin rearrangements that lead to the uptake ofSalmonella. T3SS effectors mediate the uptake of S. typhimurium intonon-phagocytic host cells, such as epithelial cells. The SPI-1 T3SS hasbeen shown to be essential for crossing the gut epithelial layer, but isdispensable for infection when bacteria are injected parenterally, forexample. SPI-1 mutants have defects in epithelial cell invasion,dramatically reducing oral virulence, but are taken up normally byphagocytic cells, such as macrophages (Kong et al. (2012) Proc. Natl.Acad. Sci. U.S.A. 109(47):19414-19419). The immunostimulatory S.typhimurium strains provided herein can be engineered with mutations inSPI-1 T3SS genes, preventing their uptake by epithelial cells, andfocusing them to immune cells such as macrophages, enhancing theanti-tumor immune response.

T3SS effectors also activate the NLRC4 inflammasome in macrophages,activating caspase-1 and leading to cell death via pyroptosis.Pyroptosis is a highly inflammatory form of programmed cell that occursmost frequently following infection with intracellular pathogens, andplays a role in the antimicrobial response. This pro-inflammatory celldeath can limit the initiation of a robust adaptive immune response bydirectly inducing the death of antigen-presenting cells (APCs), as wellas modifying the cytokine milieu to prevent the generation of memoryT-cells. SPI-1 induces pyroptosis by injecting flagellin, needle and rodproteins (PrgI/J), while the extracellular flagellin stimulates TLR5signaling. Thus, engineering the immunostimulatory bacteria herein tocontain mutations in the genes involved in pyroptosis can enhance theanti-tumor immune effect by reducing cell death in immune cells such asmacrophages.

Macrophage Pyroptosis

The macrophage NLRC4 inflammasome, which plays a role in the innateimmune and antimicrobial responses, is a large multi-protein complexthat recognizes cytosolic pathogens and provides for the autocatalyticactivation of caspase-1. Activation of caspase-1 induces maturation andrelease of the pro-inflammatory cytokines IL-1β and IL-18, and triggerspyroptosis, a rapid inflammatory form of macrophage cell death.Infection by certain Gram-negative bacteria encoding type 3 or 4secretion systems, such as Salmonella typhimurium and Pseudomonasaeruginosa, triggers the activation of the NLRC4 inflammasome uponrecognition of bacterial ligands such as needle protein, rod protein andflagellin, following translocation into the host cell cytosol by the Stmpathogenicity island-1 type III secretion system (SPI-1 T3SS).Pyroptosis is not limited to macrophages; caspase-1-dependent death hasbeen observed in dendritic cells following infection with Salmonella (Liet al. (2016) Scientific Reports 6:37447; Chen et al. (2014) CellReports 8:570-582; Fink and Cookson (2007) Cellular Microbiology9(11):2562-2570). As shown herein, the knock-out of genes in theSalmonella genome involved in the induction of pyroptosis enhances theanti-tumor immune response. This prevents the loss of immune cells,including macrophages, following bacterial infection. For example, genesencoding hilA, rod protein (PrgJ), needle protein (PrgI), flagellinand/or QseC can be knocked out/disrupted in the immunostimulatorybacteria provided herein.

hilA

The invasion-associated Salmonella pathogenicity island-1 (SPI-1),including the type 3 secretion system (T3SS), is responsible for thetranslocation of effector proteins into the cytosol of host cells,causing actin rearrangements that lead to the uptake of Salmonella. hilAis a transcriptional activator for SPI-1 genes, and its expression isregulated by environmental signals such as, for example, oxygen,osmolarity, pH and growth phase. Suboptimal conditions repress theexpression of hilA, thereby suppressing the invasive phenotype of thebacterium (Kong et al. (2012) Proc. Natl. Acad. Sci. U.S.A.109(47):19414-19419). T3SS effectors mediate the uptake of S.typhimurium into non-phagocytic host cells, such as epithelial cells.The SPI-1 T3SS is essential for crossing the gut epithelial layer, butis dispensable for infection, such as when bacteria are injectedparenterally. SPI-1 mutants have defects in epithelial cell invasion,reducing oral virulence, but are taken up normally by phagocytic cells,such as macrophages. The immunostimulatory bacteria provided hereininclude those with deletion or disruption of the hilA gene and/or othergenes in the T3SS pathway. When these bacteria are administered, such asintravenously or intratumorally, infection is focused towards phagocyticcells, such as macrophages and dendritic cells, that do not require theSPI-1 T3SS for uptake. This enhances the safety profile of theimmunostimulatory bacteria provided herein. It prevents off-target cellinvasion and prevents fecal-oral transmission.

In addition to reducing the uptake of Salmonella by non-phagocyticcells, such as epithelial cells, deletion or disruption of hilA and/orother genes in this pathway also prolongs the longevity of thephagocytic cells, by preventing pyroptosis in macrophages, thus,inducing less cell death in human macrophages compared to bacteria thatdo not contain a deletion in hilA. For example, hilA deficientSalmonella strains prevent pyroptosis by preventing inflammasomeactivation, but maintain TLR5 signaling. hilA deletion/disruption alsoallows for the prolonged secretion of cytokines, such as those encodedby the immunostimulatory bacteria provided herein, and for macrophagetrafficking to tumors, thus improving the efficacy of theimmunostimulatory bacteria. For example, in comparison to S. typhimuriumcontaining intact hilA, such as VNP20009, the hilA deletion mutants,exemplified herein, further reduce the quantity of pro-inflammatorycytokines, such as IL-6, increasing the tolerability of the therapy, aswell as the quality of the adaptive immune response.

Flagellin

Bacterial, such as Salmonella, flagellin, in addition to SPI-1 T3SS, isnecessary for triggering pyroptosis in macrophages, and can be detectedby the macrophage NLRC4 inflammasome. Flagellin, which is the majorcomponent of flagellum, is recognized by TLR5. Salmonella encodes twoflagellin genes, fliC and fljB; elimination of flagellin subunitsdecreases pyroptosis in macrophages. For example, S. typhimurium withdeletions in fliC and fljB resulted in significantly reduced IL-1βsecretion compared to the wild-type strain, whereas cellular uptake andintracellular replication of the bacterium remained unaffected. Thisdemonstrates that flagellin plays a significant role in inflammasomeactivation. Additionally, S. typhimurium strains engineered toconstitutively express FliC were found to induce macrophage pyroptosis(Li et al. (2016) Scientific Reports 6:37447; Fink and Cookson (2007)Cellular Microbiology 9(11):2562-2570; Winter et al. (2015) Infect.Immun. 83(4):1546-1555). The genome of the immunostimulatory bacteriaherein can be modified to delete or mutate the flagellin genes fliC andfljB in S. typhimurium, leading to decreased cell death of tumorresident immune cells, such as macrophages, and enhancing the antitumorimmune response of the immunostimulatory bacteria.

Rod Protein (PrgJ)

NLRC4 also detects aflagellated S. typhimurium. Theflagellin-independent response was discovered to be due to the detectionof PrgJ, which is the SPI-1 T3SS rod protein in S. typhimurium. Deliveryof purified PrgJ protein to the macrophage cytosol resulted in rapidNLRC4-dependent caspase-1 activation, as well as secretion of IL-1β,similar to the effects induced by flagellin (Miao et al. (2010) Proc.Natl. Acad. Sci. U.S.A. 107(7):3076-3080). Thus, the mutation orknockout of the gene encoding PrgJ in S. typhimurium can reducemacrophage pyroptosis, which enhances the antitumor immune effect of theimmunostimulatory bacteria, by preserving immune cells that aresusceptible to being killed by the bacteria.

Needle protein (PrgI)

PrgI, which is the SPI-1 T3SS needle protein in S. typhimurium, also isrecognized by, and activates, NLRC4. The delivery of S. typhimurium PrgIto the cytosol of human primary monocyte-derived macrophages resulted inIL-10 secretion and subsequent cell death, while a Salmonella mutantthat expresses PrgI but not flagellin was shown to activate theinflammasome in primary monocyte-derived macrophages at later timepoints than strains expressing flagellin (Kortmann et al. (2015) J.Immunol. 195:815-819). The immunostimulatory bacteria provided hereincan be modified to mutate or delete the gene encoding needle protein inS. typhimurium, preventing immune cell pyroptosis, and enhancing theantitumor immune effect.

QseC

QseC is a highly conserved membrane histidine sensor kinase that isfound in many Gram-negative bacteria, responds to the environment andregulates the expression of several virulence factors, including theflhDC gene that encodes the master regulator of flagellum biosynthesisin S. typhimurium; the sopB gene, which encodes a protein that plays arole in the invasion of non-phagocytic cells, the early maturation andregulation of trafficking of the Salmonella-containing vacuole (SCV),and the inhibition of SCV-lysosome fusion; and the sifA gene, which isrequired for SCV maintenance and membrane integrity. It has been shownthat selective inhibition of QseC by LED209 inhibits bacterial virulencewithout suppressing S. typhimurium growth, by inhibiting theQseC-mediated activation of virulence-related gene expression (e.g.,flhDC, sifA and sopB), and partially protects mice from death followinginfection with S. typhimurium or Francisella tularensis. QseC blockadewas found to inhibit caspase-1 activation, IL-1β release, and S.typhimurium-induced pyroptosis of macrophages, by inhibiting excessinflammasome activation in the infected macrophages. Inhibition of QseCalso suppressed flagellar gene expression and motility, and suppressedthe invasion and replication capacities of S. typhimurium in epithelialcells (Li et al. (2016) Scientific Reports 6:37447). Thus, modificationof the immunostimulatory bacteria herein, to mutate or knockout the geneencoding QseC, can enhance the antitumor immune response by focusing S.typhimurium infection to non-epithelial cells, and by reducing celldeath in immune cells, such as by preventing pyroptosis in macrophages.

7. Bacterial Culture Conditions

Culture conditions for bacteria can influence their gene expression. Ithas been documented that S. typhimurium can induce rapidpro-inflammatory caspase-dependent cell death of macrophages, but notepithelial cells, within 30 to 60 min of infection by a mechanisminvolving the SPI-1 and its associated T3SS-1 (Lundberg et. al (1999)Journal of Bacteriology 181(11):3433-3437). It is now known that thiscell death is mediated by activation of the inflammasome thatsubsequently activates caspase-1, which promotes the maturation andrelease of IL-10 and IL-18 and initiates a novel form of cell deathcalled pyroptosis (Broz and Monack (2011) Immunol. Rev. 243(1):174-190). This pyroptotic activity can be induced by using log phasebacteria, whereas stationary phase bacteria do not induce this rapidcell death in macrophages. The SPI-1 genes are induced during log phasegrowth. Thus, by harvesting S. typhimurium to be used therapeutically atstationary phase, rapid pyroptosis of macrophages can be prevented.Macrophages are important mediators of the innate immune system and theycan act to secrete cytokines that are critical for establishingappropriate anti-tumor responses. In addition, limiting pro-inflammatorycytokines such as IL-10 and IL-18 secretion will improve thetolerability of administered S. typhimurium therapy. As provided herein,immunostimulatory S. typhimurium harvested at stationary phase will beused to induce anti-tumor responses.

E. Bacterial Attenuation and Colonization

1. Deletion of Flagellin (fliC/Fljg)

Provided are immunostimulatory bacteria, such as the Salmonella speciesS. typhimurium, engineered to lack both flagellin subunits fliC andfljB, to reduce pro-inflammatory signaling. For example, as shownherein, a Salmonella strain lacking msbB, which results in reducedTNF-alpha induction, is combined with fliC and fljB knockouts. Theresulting Salmonella strain has a combined reduction in TNF-alphainduction and reduction in TLR5 recognition. These modifications, msbB⁻,fliC⁻ and fljB⁻, can be combined with a bacterial plasmid, optionallycontaining CpGs, and also a cDNA expression cassette to provideexpression of a therapeutic protein under the control of a eukaryoticpromoter, such as for example, an immunostimulatory protein, such as acytokine or chemokine, such as IL-2, and/or also inhibitory molecules,such as antibodies, including antibody fragments, such as nanobodies,and/or RNAi molecule(s), targeting an immune checkpoint, such as TREX1,PD-L1, VISTA, SIRP-alpha, TGF-beta, beta-catenin, CD47, VEGF, andcombinations thereof. The resulting bacteria have reducedproinflammatory signaling, and robust anti-tumor activity.

For example, as exemplified herein, a fliC⁻ and fljB⁻ double mutant wasconstructed in the asd-deleted strain of S. typhimurium strain VNP20009or in a wild-type Salmonella typhimurium, such as one having all of theidentifying characteristics of the strain deposited under ATCC accessionno. 14028. VNP20009, which is a derivative of ATCC 14028, was attenuatedfor virulence by disruption of purI/purM, and was also engineered tocontain an msbB deletion that results in production of a lipid A subunitof LPS that is less toxigenic than wild-type lipid A. This results inreduced TNF-α production in the mouse model after intravenousadministration, compared to strains with wild-type lipid A.

A fliC⁻ and fljB⁻ double mutant was constructed on a wild-type strain ofS. typhimurium and also engineered to contain the asd, purI/purM andmsbB deletions. The bacterium is optionally pagP⁻. The resulting strainsare exemplary of strains that are attenuated for bacterial inflammationby modification of lipid A to reduce TLR2/4 signaling, and deletion ofthe flagellin subunits to reduce TLR5 recognition and inflammasomeinduction. Deletion of the flagellin subunits combined with modificationof the LPS allows for greater tolerability in the host, and directs theimmunostimulatory response towards production of immunostimulatoryproteins. The delivery of RNA interference by the modified bacteriaagainst desired targets in the TME elicits an anti-tumor response andpromotes an adaptive immune response to the tumor.

2. Deletion of Genes in the LPS Biosynthetic Pathway

The LPS of Gram-negative bacteria is the major component of the outerleaflet of the bacterial membrane. It is composed of three major parts,lipid A, a nonrepeating core oligosaccharide, and the O antigen (or Opolysaccharide). O antigen is the outermost portion on LPS and serves asa protective layer against bacterial permeability, however, the sugarcomposition of O antigen varies widely between strains. The lipid A andcore oligosaccharide vary less, and are more typically conserved withinstrains of the same species. Lipid A is the portion of LPS that containsendotoxin activity. It is typically a disaccharide decorated withmultiple fatty acids. These hydrophobic fatty acid chains anchor 5 theLPS into the bacterial membrane, and the rest of the LPS projects fromthe cell surface. The lipid A domain is responsible for much of thetoxicity of Gram-negative bacteria. Typically, LPS in the blood isrecognized as a significant pathogen associated molecular pattern(PAMP), and induces a profound pro-inflammatory response. LPS is theligand for a membrane-bound receptor complex comprising CD14, MD2 andTLR4. TLR4 is a transmembrane protein that can signal through the MyD88and TRIF pathways to stimulate the NFκB pathway and result in theproduction of pro-inflammatory cytokines such as TNF-α and IL-1β, theresult of which can be endotoxic shock, which can be fatal. LPS in thecytosol of mammalian cells can bind directly to the CARD domains ofcaspases 4, 5, and 11, leading to autoactivation and pyroptotic celldeath (Hagar et al. (2015) Cell Research 25:149-150). The composition oflipid A and the toxigenicity of lipid A variants is well documented. Forexample, a monophosphorylated lipid A is much less inflammatory thanlipid A with multiple phosphate groups. The number and length of theacyl chains on lipid A can also have a profound impact on the degree oftoxicity. Canonical lipid A from E. coli has six acyl chains, and thishexa-acylation is potently toxic. S. typhimurium lipid A is similar tothat of E. coli; it is a glucosamine disaccharide that carries fourprimary and two secondary hydroxyacyl chains (Raetz and Whitfield (2002)Annu. Rev. Biochem. 71:635-700). As described above, msbB⁻ mutants of S.typhimurium cannot undergo the terminal myristoylation of its LPS andproduce predominantly penta-acylated lipid A that is significantly lesstoxic than hexa-acylated lipid A. The modification of lipid A withpalmitate is catalyzed by palmitoyl transferase (PagP). Transcription ofthe pagP gene is under control of the PhoP/PhoQ system, which isactivated by low concentrations of magnesium, e.g., inside the SCV.Thus, the acyl content of S. typhimurium is variable, and with wild-typebacteria, it can be hexa- or penta-acylated. The ability of S.typhimurium to palmitate its lipid A increases resistance toantimicrobial peptides that are secreted into phagolysozomes.

In wild-type S. typhimurium, expression of pagP results in a lipid Athat is hepta-acylated. In an msbB⁻ mutant (in which the terminal acylchain of the lipid A cannot be added), the induction of pagP results ina hexa-acylated LPS (Kong et al. (2011) Infection and Immunity79(12):5027-5038). Hexa-acylated LPS has been shown to be the mostpro-inflammatory. While other groups have sought to exploit thispro-inflammatory signal, for example, by deletion of pagP to allow onlyhexa-acylated LPS to be produced (Felgner et al. (2016) Gut Microbes7(2): 171-177; Felgner et al. (2018) Oncoimmunology 7(2): e1382791),this can lead to poor tolerability, due to the TNF-α-mediatedpro-inflammatory nature of the LPS and paradoxically less adaptiveimmunity (Kocijancic et al. (2017) Oncotarget 8(30):49988-50001).Exemplified herein, is a live attenuated strain of S. typhimurium thatcan only produce penta-acylated LPS, that contains a deletion of themsbB gene (that prevents the terminal myristoylation of lipid A, asdescribed above), and is further modified by deletion of pagP(preventing palmitoylation). A strain modified to produce penta-acylatedLPS will allow for lower levels of pro-inflammatory cytokines, improvedstability in the blood and resistance to complement fixation, increasedsensitivity to antimicrobial peptides, enhanced tolerability, andincreased anti-tumor immunity when further modified to expressheterologous immune-stimulatory proteins and/or interfering RNAs againstimmune checkpoints.

As provided herein, a pagP⁻ mutant was also constructed on an asd, msbB,purI/purM and fliC/fljB deleted strain of S. typhimurium VNP20009 orwild-type S. typhimurium. The resulting strains are exemplary of strainsthat are attenuated for bacterial inflammation by modification of lipidA to reduce TLR2/4 signaling, and deletion of the flagellin subunits toreduce TLR5 recognition and inflammasome induction, and deletion of pagPto produce penta-acylated LPS. Deletion of the flagellin subunitscombined with modification of the LPS allows for greater tolerability inthe host, and greater stability in the blood and resistance tocomplement fixation, providing for improved trafficking to the tumorsite, in order to direct the immuno-stimulatory response towardsproduction of any gene product, such as immune-stimulatory proteinsand/or delivery of RNA interference against desired targets in the TMEto elicit an anti-tumor response and promote an adaptive immune responseto the tumor.

3. Colonization

VNP20009 is an attenuated S. typhimurium-based microbial cancer therapythat was developed for the treatment of cancer. VNP20009 is attenuatedthrough deletion of the genes msbB and purI⁻ (purM⁻). The purI⁻ deletionrenders the microbe auxotrophic for purines or adenosine. Deletion ofthe msbB gene reduced the toxicity associated with lipopolysaccharide(LPS) by preventing the addition of a terminal myristyl group to thelipid A domain (Kahn et al., (1998) Mol. Microbiol. 29:571-579).

There is a difference between mouse and humans in the ability ofVNP20009 to colonize tumors. Systemic administration of VNP20009resulted in colonization of mouse tumors; whereas systemicadministration of VNP20009 in human patients resulted in very littlecolonization. It was shown that in mice, VNP20009 showed a high degreeof tumor colonization after systemic administration (Clairmont et al.,(2000) J Infect Dis. 181:1996-2002; and Bermudes et al. (2001)Biotechnol. Genet. Eng. Rev. 18:219-33). In a Phase 1 Study in advancedmelanoma patients, however, very little VNP20009 was detected in humantumors after a 30-minute intravenous infusion (see Toso et al., (2002)J. Clin. Oncol. 20:142-52). Patients that entered into a follow-up studyevaluating a longer, four-hour infusion of VNP20009, also demonstrated alack of detectable VNP20009 after tumor biopsy (Heimann et al. (2003) J.Immunother. 26:179-180). Following intratumoral administration,colonization of a derivative of VNP20009 was detected (Nemunaitis et al.(2003) Cancer Gene Ther. 10:737-44). Direct intratumoral administrationof VNP20009 to human tumors resulted in tumor colonization, indicatingthat human tumors can be colonized at a high level, and that thedifference in tumor colonization between mice and humans occurs onlyafter systemic administration.

It is shown herein (see, e.g., Example 25) that VNP20009 is inactivatedby human complement, which leads to low tumor colonization. Strains thatprovide improved resistance to complement are provided. These strainscontain modifications in the bacterial genome and also can carry aplasmid, typically in low or medium copy number, to encode genes toprovide for replication (asd under the control of a eukaryoticpromoter), and nucleic acid(s) encoding a therapeutic product(s), suchas, but not limited to, RNAi, immunostimulatory protein, such ascytokines, and other such therapeutic genes, as described elsewhereherein. The table below summarizes the bacterialgenotypes/modifications, their functional effects, and theeffects/benefits.

Genotype/Modification Functional effect Effect/Benefit ΔpurIPurine/adenosine Tumor-specific enrichment auxotrophy Limitedreplication in healthy tissue ΔmsbB LPS surface coat Decreased TLR4recognition modification Reduced cytokine profile Improved safety ΔFLGFlagella knockout Removes major inflammatory and immune-suppressiveelement Decreased TLR5 recognition Reduced cytokine profile Improvedsafety ΔpagP LPS surface coat Removes major inflammatory andmodifications immune-suppressive element Decreased TLR4 recognitionReduced IL-6 profile Improved safety Δasd (in genome) Plasmidmaintenance Improved plasmid delivery Plasmid maintenance plasmidExpress gene Eukaryotic promoter limits expression products under tocells containing the plasmid control of host- Long term expression inthe TME recognized promoter (i.e., asd encoded on plasmid under controlof host-recognized promoter) Expression of therapeutic product(s)

Strains provided herein are ΔFLG and/or ΔpagP. Additionally, the strainsare one or more of ΔpurI (ΔpurM), ΔmsbB, and Δasd (in the bacterialgenome). The plasmid is modified to encode products under control ofhost-recognized promoters (e.g., eukaryotic promoters, such as RNApolymerase II promoters, including those from eukaryotes, and animalviruses). The plasmids can encode asd to permit replication in vivo, aswell as nucleic acids with other beneficial functions and gene productsas described elsewhere herein.

The immunostimulatory bacteria are derived from suitable bacterialstrains. Bacterial strains can be attenuated strains, or strains thatare attenuated by standard methods, or that, by virtue of themodifications provided herein, are attenuated in that their ability tocolonize is limited primarily to immunoprivileged tissues and organs,particularly immune and tumor cells, including solid tumors. Bacteriainclude, but are not limited to, for example, strains of Salmonella,Shigella, Listeria, E. coli, and Bifidobacteriae. For example, speciesinclude Shigella sonnei, Shigella flexneri, Shigella disenteriae,Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium,Salmonella gallinarum, and Salmonella enteritidis. Other suitablebacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria,Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia,Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella,Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, andErysipelothrix. For example, Rickettsia Rikettsiae, Rickettsiaprowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsiasibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseriagonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisellatularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii,Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus,Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi,Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillussubtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica,Rochalimaea quintana, and Agrobacterium tumerfacium.

Exemplary of the immunostimulatory bacteria provided herein are speciesof Salmonella. Exemplary of bacteria for modification as describedherein are wild-type strains of Salmonella, such as the strain that hasall of the identifying characteristics of the strain deposited in theATCC as accession #14028. Engineered strains of Salmonella typhimurium,such as strain YS1646 (ATCC Catalog #202165; also referred to asVNP20009, see, International Application Publication No. WO 99/13053,)that is engineered with plasmids to complement an asd gene knockout andantibiotic-free plasmid maintenance. The strains then are modified todelete the flagellin genes and/or to delete pagP. The strains also arerendered auxotrophic for purines, particularly adenosine, and are asd⁻and msbB⁻. The asd gene can be provided on a plasmid for replication inthe eukaryotic host. These deletions and plasmids are describedelsewhere herein. Any of the nucleic acid encoding therapeutic productsand immunostimulatory proteins and products, described elsewhere hereinand/or known to those of skill in the art, can be included on theplasmid. The plasmid generally is present in low to medium copy numberas described elsewhere herein. Therapeutic products includeimmunostimulatory proteins, such as cytokines, that promote ananti-tumor immune response in the tumor microenvironment and other suchproducts described herein.

F. Constructing Exemplary Plasmids Encoding Therapeutic Proteins

The immunostimulatory bacteria provided herein are modified. Theyinclude modifications to the bacterial genome and to bacterialexpression and host cell invasion, as discussed below, such as toimprove or increase targeting to or accumulation in tumors,tumor-resident immune cells, and the tumor microenvironment, and also,to include plasmids that encode products that are expressed in thebacteria by including a bacterial promoter, or in the host by includingan appropriate eukaryotic promoter and other regulatory regions asappropriate. It is shown herein that the immunostimulatory bacteria thatare flagellin⁻ (fliC⁻/fljB⁻) and/or pagP⁻, and optionally hilA⁻, exhibitincreased tumor colonization, and, thus, can overcome the previousproblems encountered with VNP20009, which failed to adequately colonizetumors in humans. The clinical activity of VNP20009 was disappointing inpart due to its poor ability to colonize human tumors (Nemunaitis et al.(2003) Cancer Gene Ther. 10(10):737-744; Toso et al. (2002) J. Clin.Oncol. 20(1): 142-152; Heimann et al. (2003) J. Immunother. 26(2):179-180).

To introduce the plasmids, the bacteria are transformed using standardmethods, such as electroporation, with purified DNA plasmids constructedwith routine molecular biology tools and methods (DNA synthesis, PCRamplification, DNA restriction enzyme digestion and ligation ofcompatible cohesive end fragments with ligase). As discussed throughout,the plasmids encode one or more therapeutic products, includingproteins, such as antibodies and fragments thereof, andimmunostimulatory proteins, such as interleukins, under control ofhost-recognized promoters. The encoded immunostimulatory proteinsstimulate the immune system, particularly in the tumor microenvironment.The antibodies, including antibody fragments and single chainantibodies, can inhibit immune checkpoints.

The plasmid can encode, for example, a therapeutic product ortherapeutic protein that is one or more of GM-CSF, IL-2, IL-7, IL-12p70(IL-12p40+IL-12p35), IL-15, IL-15/IL-15R alpha chain complex, IL-2 thathas attenuated binding to IL-2Ra, IL-18, IL-36 gamma, CXCL9, CXCL10,CXCL11, CCL3, CCL4, CCL5, proteins that are involved in or that effector potentiate recruitment/persistence of T cells, CD40, CD40 ligand,OX40, OX40 ligand, 4-1BB, 4-1BB ligand, members of the B7-CD28 family, aTGF-beta polypeptide antagonist, a CD47 antagonist, interferon-α,interferon-β, interferon-γ, or members of the tumor necrosis factorreceptor (TNFR) superfamily.

The bacteria can encode other products on the plasmids, such as one ormore short hairpin (sh) RNA construct(s), or other inhibitory RNAmodalities, whose expression inhibits, suppresses or disrupts expressionof targeted genes. The therapeutic products, such as theimmunostimulatory proteins, antibodies, and RNAi, such as shRNA ormicroRNA constructs, are expressed under control of a eukaryoticpromoter, such as an RNA polymerase (RNAP) II or III promoter.Typically, RNAPIII (also referred to as POLIII) promoters areconstitutive, and RNAPII (also referred to as POLII) can be regulated.In some examples, the shRNAs target the gene TREX1, to inhibit itsexpression. In some embodiments the plasmids encode a plurality oftherapeutic products. Where a plurality of products, such as RNAi's, areencoded, expression of each can be under control of different promoters,or the products can be encoded polycistronically.

The nucleic acids encoding the therapeutic products/proteins can beunder the control of a eukaryotic promoter that is an RNA polymerase IIpromoter or an RNA polymerase III promoter. The RNA polymerase IIpromoter can be a viral promoter or a mammalian RNA polymerase IIpromoter. The viral promoter can be one selected from among well-knownviral promoters. Exemplary of such are a cytomegalovirus (CMV) promoter,an SV40 promoter, an Epstein Barr virus (EBV) promoter, a herpes viruspromoter, and an adenovirus promoter.

The therapeutic product can be under the control of a eukaryotic RNApolymerase II (RNAP II) promoter. Many such promoters are very wellknown. Exemplary of such promoters is an RNAPII promoter selected fromamong, for example, an elongation factor-1 (EF1) alpha promoter, aubiquitin C (UBC) promoter, a phosphoglycerate kinase 1 promoter (PGK)promoter, a CAG promoter (which consists of: (C) the cytomegalovirus(CMV) early enhancer element, (A) the promoter, the first exon and thefirst intron of chicken beta-actin gene, and (G) the splice acceptor ofthe rabbit beta-globin gene), an EIF4a1 (eukaryotic initiation factor4A) promoter, a CBA promoter (chicken beta actin), an MND promoter, aGAPDH promoter, and a CD68 promoter. MND is a synthetic promoter thatcontains the U3 region of a modified MoMuLV LTR with myeloproliferativesarcoma virus enhancer (murine leukemia virus-derived MND promoter(myeloproliferative sarcoma virus enhancer, negative control regiondeleted, d1587rev primer-binding site substituted; see, e.g., Li et al.(2010)J. Neurosci. Methods vol. 189:56-64).

As provided herein, bacterial strains, such as strains of Salmonella,including S. typhimurium, are modified or identified to be auxotrophicfor adenosine in the tumor microenvironment, and/or the genome of theimmunostimulatory bacterium is modified so that it preferentiallyinfects tumor-resident immune cells and/or so that it induces less celldeath in tumor-resident immune cells, and the bacteria are modified tocarry plasmids encoding a therapeutic product, such as animmunostimulatory protein, an antibody or antibody fragment, such as ananti-tumor antibody or fragment thereof or anti-checkpoint antibody, andother products, such as RNAi.

1. Immunostimulatory Proteins

As discussed below, and elsewhere herein, provided are immunostimulatorybacteria that contain sequences of nucleotides that encode geneproducts, such as immunostimulatory proteins, to confer, increase, orenhance immune responses in the tumor microenvironment. Theseimmunostimulatory bacteria are modified to preferentially infect tumors,including tumor-resident immune cells, and/or the genome of theimmunostimulatory bacteria is modified so that they induce less celldeath in tumor-resident immune cells, whereby the immunostimulatorybacteria accumulate in tumor cells to thereby deliver theimmunostimulatory proteins to the targeted cells to stimulate the immuneresponse against the tumor. The immunostimulatory bacteria can furtherencode a tumor antigen to enhance the response against the particulartumor. Any of the immunostimulatory bacteria provided herein anddescribed above and below can be modified to encode an immunostimulatoryprotein. Generally, the immunostimulatory protein is under the controlof an RNA polymerase II (RNAPII) promoter, and also is encoded in theplasmid for secretion, upon expression, into the tumor microenvironment.Any of the bacteria described herein for modification, such as any ofthe strains of Salmonella, Shigella, E. coli, Bifidobacteriae,Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria,Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter,Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella,Rhodococcus, Pseudomonas, Helicobacter, Bacillus, and Erysipelothrix, oran attenuated strain thereof or a modified strain thereof encode theimmunostimulatory protein so that it is expressed in the infectedsubject's cells. The immunostimulatory bacteria include those that aremodified, as described herein, to colonize, accumulate in, or topreferentially infect, tumors, tumor-resident immune cells and/or theTME.

As discussed in section D5, and elsewhere herein, the immunostimulatorybacteria can encode immunostimulatory proteins, such as cytokines,including chemokines, that enhance or stimulate or evoke an anti-tumorimmune response, particularly when expressed in tumors, in the tumormicroenvironment and/or in tumor-resident immune cells.

The immunostimulatory bacteria herein can be modified to encode animmunostimulatory protein that promotes or induces or enhances ananti-tumor response. The immunostimulatory protein can be encoded on aplasmid in the bacterium, under the control of a eukaryotic promoter,such as a promoter recognized by RNA polymerase II, for expression in aeukaryotic subject, particularly the subject for whom theimmunostimulatory bacterium is to be administered, such as a human. Thenucleic acid encoding the immunostimulatory protein can include, inaddition to the eukaryotic promoter, other regulatory signals forexpression or trafficking in the cells, such as for secretion orexpression on the surface of a cell.

Immunostimulatory proteins are those that, in the appropriateenvironment, such as a tumor microenvironment (TME), can promote orparticipate in or enhance an anti-tumor response by the subject to whomthe immunostimulatory bacterium is administered. Immunostimulatoryproteins include, but are not limited to, cytokines, chemokines andco-stimulatory molecules. These include cytokines, such as, but notlimited to, IL-2, IL-7, IL-12, IL-15, and IL-18; chemokines, such as,but not limited to, CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11; and/orco-stimulatory molecules, such as, but not limited to, CD40, CD40L,OX40, OX40L, 4-1BB, 4-1BBL, GM-CSF, members of the TNF/TNFR superfamilyand members of the B7-CD28 family. Other such immunostimulatory proteinsthat are used for treatment of tumors or that can promote, enhance orotherwise increase or evoke an anti-tumor response, known to those ofskill in the art, are contemplated for encoding in the immunostimulatorybacteria provided herein.

In some embodiments, the immunostimulatory bacteria herein areengineered to express cytokines to stimulate the immune system,including, but not limited to, IL-2, IL-7, IL-12 (IL-12p70 (IL12p40+IL-12p35)), IL-15 (and the IL-15:IL-15R alpha chain complex), andIL-18. Cytokines stimulate immune effector cells and stromal cells atthe tumor site, and enhance tumor cell recognition by cytotoxic cells.In some embodiments, the immunostimulatory bacteria can be engineered toexpress chemokines, such as, for example, CCL3, CCL4, CCL5, CXCL9,CXCL10 and CXCL11. These modifications and bacteria encoding them arediscussed above, and exemplified below.

2. Antibodies and Antibody Fragments

Provided are immunostimulatory bacteria that contain sequences ofnucleotides that encode gene products, such as antibodies and antibodyfragments, to confer, increase, or enhance anti-tumor immune responses.These include antibodies and antibody fragments that target immunecheckpoints, such as CTLA-4, PD-1, PD-L1, CD47, or that target tumorantigens and tumor neoantigens, including those identified from thetumor of a subject to be treated, amongst others, and, for example,anti-IL-6 antibodies that modulate, particularly inhibit, immunesuppression. The antibodies or fragments thereof, such as scFv and othersingle chain antibodies, such as camelids and nanobodies, can be encodedon a plasmid in the bacterium, under the control of eukaryoticregulatory sequences and signals, including a eukaryotic promoter, suchas a promoter recognized by RNA polymerase II, for expression in aeukaryotic subject, particularly the subject for whom theimmunostimulatory bacterium is to be administered, such as a human. Thenucleic acid encoding the antibodies and antibody fragments can include,in addition to the eukaryotic promoter, other regulatory signals forexpression and/or trafficking in the cells, such as for secretion orexpression on the surface of a cell.

These immunostimulatory bacteria are those provided herein whose genomesare modified to preferentially infect tumors, including tumor-residentimmune cells, and/or to eliminate infection of cells that are not targetcells, and/or so that they induce less cell death in tumor-residentimmune cells, whereby the immunostimulatory bacteria accumulate in tumorcells to thereby deliver the antibody or antibody fragment to thetargeted cells to stimulate the immune response against the tumor. Theimmunostimulatory bacteria also or alternatively can encode a tumorantigen or neoantigen to enhance the response against the particulartumor. Any of the immunostimulatory bacteria provided herein anddescribed above and below can be modified to encode an antibody orfragment thereof. Generally, the antibody or fragment thereof is underthe control of an RNA polymerase II (RNAPII) promoter, and also isencoded in the plasmid for secretion, upon expression, into the tumormicroenvironment. Any of the bacteria described herein for modification,such as any of the strains of Salmonella, Shigella, E. coli,Bifidobacteriae, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella,Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium,Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium,Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter,Bacillus, and Erysipelothrix, or an attenuated strain thereof or amodified strain thereof encode the antibody or fragment thereof so thatit is expressed in the infected subject's cells. The immunostimulatorybacteria include those that are modified, as described herein, tocolonize, accumulate in, or to preferentially infect, tumors,tumor-resident immune cells and/or the tumor microenvironment.

Therapeutic antibodies and fragments thereof are well known. Forexample, there are a plethora of anti-CTLA4, anti-PD-1 and anti-PD-L1antibodies and antigen-binding fragments thereof, such as Fab, Fab′,F(ab′)₂, single-chain Fv (scFv), Fv, disulfide-stabilized Fv (dsFv),nanobodies and camelids, and diabody fragments, single-chain antibodies,and humanized and human antibodies that are known. For example,antibodies that bind to PD-1 or PD-L1 and inhibit PD-1-inhibitoryactivity, and that have been used in anti-tumor immunotherapy are known.Exemplary anti-PD-1 antibodies include, but are not limited to, any ofthose described in U.S. Pat. Nos. 7,943,743, 8,008,449, and 8,735,553;U.S. Publication Nos. 2005/0180969 and 2007/0166281; and InternationalPatent Application Publication No. WO 2008/156712. Anti-PD-L1 antibodiesinclude, but are not limited to, any of those described in U.S. Publ.Nos. 2013/0034559 and 2013/0045202; U.S. Pat. Nos. 7,943,743, 8,217,149,8,679,767, and 8,779,108; and Intl. App. Publ. Nos. WO 2010/077634 andWO 2013/019906. Several antibodies, which bind to and inhibit CTLA-4activity, have been described, and have been used in anti-tumorimmunotherapy. Anti-CTLA4 antibodies include, but are not limited to,any of those described in U.S. Pat. Nos. 6,682,736 and 6,984,720; U.S.Publ. Nos. 2002/0086014 and 2009/0074787; European Patent No. EP1262193; and International Patent Application Publication No. WO2000/037504. Anti-CTLA4 antibodies include Ipilimumab (also calledMDX-010 or 10D1) and Tremelimumab (also called Ticilimumab orCP-675,206). These anti-CTLA4 antibodies have been involved in numerousclinical trials for the treatment of cancers. Ipilimumab is FDA approvedfor the treatment of melanoma and has been in clinical trials for othercancers, such as prostate cancer, lung cancer, and renal cell carcinoma(RCC). Tremelimumab has been investigated in clinical trials for thetreatment of colorectal cancer (CRC), gastric cancer, melanoma andnon-small cell lung cancer (NSCLC). Exemplary checkpoint inhibitorsinclude, but are not limited to, anti-CTLA4 agents, anti-PD-1 agents,anti-PD-L1 agents and others, exemplary of which are the following:

Exemplary immune checkpoint target proteins and inhibitors TargetAntibody/fusion Target Function protein Synonyms and Code Names CTLA4Inhibitory Ipilimumab (MDX-CTLA-4; BMS-734016; MDX-010) receptorTremelimumab (Ticilimumab; CP-675, 206) PD-1 Inhibitory MK-3475(Pembrolizumab; Lambrolizumab; SCH 900475) receptor AMP-224 (anti-PD-1fusion protein AMP-224) Nivolumab (BMS-936558; MDX-1106; ONO-4538)Pidilizumab (CT-011) PD-L1 Ligand for MDX-1105 (RG7446) PD-1 BMS-936559MED14736 MPDL33280A

The immunostimulatory bacteria provided herein can be engineered toexpress any antibody/antigen-binding fragment thereof, including, butnot limited to the anti-checkpoint antibodies (checkpointantagonists/inhibitors) described herein, and known to those of skill inthe art.

3. Interfering RNAs (RNAi)

The plasmids in the immunostimulatory bacterial strains herein encodethe RNAi nucleic acids targeting the immune checkpoints and othertargets of interest, as described above. RNAi includes shRNA, siRNA, andmicroRNA. RNA interference (RNAi) allows for the sequence-selectivesuppression of gene expression in eukaryotic cells using smallinterfering RNAs (siRNAs), which are short, synthetic, dsRNA moleculeswith a sequence homologous to the target gene. RNAi technology providesa powerful tool for the depletion of disease-related transcripts.

a. shRNA

The siRNAs, which are typically about 19-29 base pairs long, function bydegrading specific host mRNA sequences, precluding translation intotheir respective protein products, effectively silencing the expressionof the target gene. Short hairpin RNAs (shRNAs), containing a tighthairpin loop, are widely used in RNAi. shRNAs contain of twocomplementary RNA sequences, each 19-29 bps long, linked by a loopspacer of 4-15 nucleotides. The RNA sequence that is complementary tothe target gene sequence (and is thus identical to the mRNA sequence),is known as the “sense” strand, while the strand which is complementaryto the mRNA (and identical to the target gene sequence) is known as the“antisense” or “guide” strand. shRNA transcripts are processed by anRNase III enzyme known as Dicer into siRNA duplexes. The product is thenloaded into the RNA-induced silencing complex (RISC) with Argonaute(Ago) proteins and other RNA-binding proteins. RISC then localizes theantisense, or “guide” strand to its complimentary mRNA sequence, whichis subsequently cleaved by Ago (U.S. Pat. No. 9,624,494). The use ofshRNA is preferred over siRNA, because it is more cost effective, highintracellular concentrations of siRNA are associated with off-targeteffects, and because the concentration of siRNA becomes diluted uponcell division. The use of shRNA, on the other hand, results in stable,long-term gene knockdown, without the need for multiple rounds oftransfection (Moore et al. (2010) Methods Mol. Bio. 629:141-158).

Targets of interest for RNAi, such as micro-RNA and siRNA/shRNA-mediatedsilencing include, but are not limited to, developmental genes such ascytokines and their receptors, cyclin kinase inhibitors,neurotransmitters and their receptors, growth/differentiation factorsand their receptors; oncogenes such as BCL2, ERBA, ERBB, JUN, KRAS, MYB,MYC; tumor suppressor genes such as BRCA1, BRCA2, MCC, p53; and enzymessuch as ACC synthases and oxidases, ATPases, alcohol dehydrogenases,amylases, catalases, DNA polymerases, RNA polymerases, kinases, lactasesand lipases (U.S. Pat. Nos. 7,732,417, 8,829,254, 8,383,599, 8,426,675,9,624,494; U.S. Patent Publication No. 2012/0009153). Of particularinterest are immune checkpoint targets, such as PD-1, PD-2, PD-L1,PD-L2, CTLA-4, IDO 1 and 2, CTNNB1 (β-catenin), SIRPα, VISTA, RNase H2,DNase II, CLEVER-1/Stabilin-1, LIGHT, HVEM, LAG3, TIM3, TIGIT,Galectin-9, KIR, GITR, TIM1, TIM4, CEACAM1, CD27, CD47, CD40, CD40L,CD48, CD70, CD80, CD86, CD112, CD137(4-1BB), CD155, CD160, CD200, CD226,CD244 (2B4), CD272 (BTLA), B7-H2, B7-H3, B7-H4, B7-H6, ICOS, A2aR, A2bR,HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2/4, OX40 and OX-40L.Other targets include MDR1, Arginase1, iNOs, IL-10, TGF-β, pGE2, STAT3,VEGF, VEGFR, KSP, HER2, Ras, EZH2, NIPP1, PP1, TAK1 and PLK1 (U.S.Patent Publication Nos. 2008/091375, 2009/0208534, 2014/0186401,2016/0184456, 2016/0369282; International Application Publication Nos.WO 2012/149364, WO 2015/002969, WO 2015/032165, WO 2016/025582).

Expressed RNAi, such as shRNAs, mediate long-term, stable knockdown oftheir target transcripts for as long as the shRNAs are transcribed. RNAPol II and III promoters are used to drive expression of shRNAconstructs, depending on the type of expression required. Consistentwith their normal cellular roles in producing abundant, endogenous smallRNAs, Pol III promoters (such as U6 or H1) drive high levels ofconstitutive shRNA expression, and their transcription initiation pointsand termination signals (4-6 thymidines) are well defined. Pol IIpromoter-driven shRNAs can be expressed tissue-specifically and aretranscribed as longer precursors that mimic pri-miRNAs and have cap andpolyA signals that must be processed. Such artificial miRNAs/shRNAs areefficiently incorporated into RISC, contributing to a more potentinhibition of target-gene expression; this allows lower levels of shRNAexpression and might prevent saturation of components in the RNAipathway. An additional advantage of Pol II promoters is that a singletranscript can simultaneously express several miRNA and mimic shRNAs.This multiplexing strategy can be used to simultaneously knock down theexpression of two or more therapeutic targets, or to target severalsites in a single gene product (see, e.g., U.S. Publication No.2009/0208534).

b. MicroRNA

MicroRNAs (miRNAs) are short, non-coding single-stranded RNA moleculesthat are about or are 20-24 nucleotides long. Naturally-occurring miRNAsare involved in the post-transcriptional regulation of gene expression;miRNAs do not encode genes. miRNAs have been shown to regulate cellproliferation and survival, as well as cellular differentiation. miRNAsinhibit translation or promote RNA degradation by binding to targetmRNAs that share sequence complementarity. They affect the stability andtranslation of mRNAs; miRNAs inhibit translation, and/or promote RNAdegradation, by binding to target mRNAs that share sequencecomplementarity. miRNAs, which occur in eukaryotes, are transcribed byRNA Pol II into capped and polyadenylated hairpin-containing primarytranscripts, known as primary miRNAs, or pri-miRNAs. These pri-miRNAsare cleaved by the enzyme Drosha ribonuclease III and its cofactorPasha/DGCR8 into ˜70 nucleotide long precursor miRNA hairpins, known asprecursor miRNAs, or pre-miRNAs, which are then transported from thenucleus into the cytoplasm, and cleaved by Dicer ribonuclease III intothe miRNA: miRNA* duplex, with sense and antisense strand products thatare approximately 22 nucleotides long. The mature miRNA is incorporatedinto the RNA-induced silencing complex (RISC), which recognizes andbinds target mRNAs, usually at the 3′-untranslated region (UTR), throughimperfect base pairing with the miRNA, resulting in the inhibition oftranslation, or destabilization/degradation of the target mRNA (see,e.g., Auyeung et al. (2013) Cell 152(4):844-85).

As described herein, regulating gene expression by RNA interference(RNAi), often uses short hairpin RNAs (shRNAs) to inhibit, disrupt orother interfere with expression of targeted genes. While advantageouslyused, and used herein, in some instances, shRNAs can be poor substratesfor small RNA biogenesis factors, they can be processed into aheterogeneous mix of small RNAs, and their precursor transcripts canaccumulate in cells, resulting in the induction of sequence-independent,non-specific effects and leading to in vivo toxicity. miRNAs arecontemplated for use herein. miRNA-like scaffolds, or artificial miRNAs(amiRNAs) can be used to reduce sequence-independent non-specificeffects (Watanabe et al. (2016) RNA Biology 13(1):25-33; Fellmann et al.(2013) Cell Reports 5:1704-1713). In addition to improved safetyprofiles, amiRNAs are more readily transcribed by Pol II than shRNAs,allowing for regulated and cell-specific expression. Artificial miRNAs(amiRNAs), in comparison to shRNAs, can effectively, and in some cases,more potently, silence gene expression without generating large amountsof inhibitory RNAs (McBride et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105(15):5868-5873). This effect was determined to be due to the moreeffective processing of siRNA from pre-miRNA precursors than from shRNAtranscripts (Boden et al. (2004) Nucl. Acid Res. 32(3):1154-1158).

miRNAs have been shown to regulate several cellular processes, includingcell proliferation and survival, intracellular signaling, cellularmetabolism, and cellular differentiation. In 1993, the first miRNA wasidentified in C. elegans (Lee et al. (1993) Cell 75:843-854), and later,mammalian miRNAs were identified (Pasquinelli et al. (2000) Nature408(6808):86-89). More than 17,000 miRNAs in 142 species have beenidentified, with more than 1900 miRNAs identified in humans, many ofwhich have been associated with a variety of diseases, including cancer(e.g., miR-15 and miR-16 in B-CLL, miR-125b, miR-145, miR-21, miR-155and miR-210 in breast cancer, miR-155 and let-7a in lung cancer, miR-145in gastric cancer, miR-29b in liver cancer); viral infections (e.g.,miR-122 and miR-155 in HCV infection, mir-28, miR-125b, miR-150, miR-223and miR-382 in HIV-1 infection, miR-21 and miR-223 in influenza virusinfection); immune-related diseases (e.g., miR-145, miR-34a, miR-155 andmiR-326 in multiple sclerosis, miR-146a in systemic lupus erythematosus,miR-144, miR-146a, miR-150, miR-182, miR-103 and miR-107 in type IIdiabetes, miR-200a, miR-200b, miR-429, miR-122, miR-451 and miR-27 innonalcoholic fatty liver disease, miR-29c, miR-34a, miR-155 and miR-200bin non-alcoholic steatohepatitis); and neurodegenerative diseases (e.g.,miR-30b, miR-30c, miR-26a, miR-133b, miR-184* and let-7 in Parkinson'sdisease, miR-29b-1, miR-29a and miR-9 in Alzheimer's disease) (Li andKowdley (2012) Genomics Proteomics Bioinformatics 10:246-253).

Studies have shown that specific endogenous miRNAs are up-regulated ordown-regulated in certain cancers. For example, miR-140 isdown-regulated in non-small cell lung cancer (NSCLC) and itsoverexpression was found to suppress PD-L1 (Xie et al. (2018) CellPhysiol. Biochem. 46:654-663); miR-197 is downregulated inplatinum-based chemotherapy resistant NSCLC, resulting inchemoresistance, tumorigenicity and metastasis (Fujita et al. (2015)Mol. Ther. 23(4):717-727); and several miRNAs have been found to bedown-regulated in cancer cells to allow PD-L1 expression, includingmiR-200, miR-34a and miR-138 (Yee et al. (2017) JI Biol. Chem.292(50):20683-20693). Several miRNAs also are upregulated, for examplemiR-21, miR-17 and miR-221 in lung cancer (Xie et al. (2018) CellPhysiol. Biochem. 46:654-663).

MicroRNA-103 (miR-103) was identified as the most upregulated microRNAin endothelial cells as a result of genotoxic stress and DNA damagefollowing radiation. It was found that miR-103 led to the downregulationof the TREX1, TREX2 and FANCF genes, and the decrease in TREX1expression was identified as the major mechanism by which miR-103mediates cell death and suppresses angiogenesis (Wilson et al. (2016)Nature Communications 7:13597). Since the loss of TREX1 results in theaccumulation of dsDNA and ssDNA, defective DNA repair, and release ofcytokines, miR-103 expression significantly upregulates thepro-inflammatory chemokines IP-10, RANTES, MIG, and the cytokines IL-15,IL-12 and IFN-γ, and this upregulation was due to a miR-103 mediateddecrease in TREX1 levels. Studies also revealed a significant increasein costimulatory receptors CD40 and CD160, and a decrease in the numbersof PD-L1⁺ macrophages and neutrophils in the 4T1 tumors. miR-103regulation of TREX1 is therefore a potent modulator of the immune TME.Other miRNAs that target TREX1 include miR-107 (U.S. Pat. No.9,242,000), miR-27a and miR-148b (U.S. Pat. No. 8,580,757). miRNA-103can be used in the plasmids herein to inhibit TREX1.

Artificial miRNAs (amiRNAs) can be delivered to cells and used tosilence target genes by creating a microRNA-based siRNA or shRNA vector(shRNAmir). The miR-30a backbone is often used in mammals, andapproximately 200-300 bases of the primary miRNA transcript are includedin the vector, with the miRNA hairpin placed at the center of thefragment, and the natural miRNA stem sequence being replaced with thesiRNA/shRNA-encoding sequence of interest. Viral promoters, such as CMV,MSCV and TLR promoters; cellular promoters, such as EIF-1a; induciblechimeric promoters, such as tet-CMV; and tissue-specific promoters, canbe used (Chang et al. (2013) Cold Spring Harb. Protoc.;doi:10.1101/pdb.prot075853). Other miRNAs that can be used includemir-16-2 (Watanabe et al. (2016) RNA Biology 13(1):25-33), miR-155(Chung et al. (2006) Nuc. Acids Res. 34:e53), miR17-92 (Liu et al.(2008) Nuc. Acids Res. 36(9):2811-2824), miR-15a, miR-16, miR-19b,miR-20, miR-23a, miR-27b, miR-29a, miR-30b, miR-30c, miR-104, miR-132s,miR-181, miR-191, miR-223 (U.S. Pat. No. 8,426,675), and Let-7 miRNA (WO2009/006450; WO 2015/032165).

shRNAmirs are limited by the low effectiveness ofcomputationally-predicted shRNA sequences, particularly when expressedunder low or single copy conditions. Third generation artificial miRNAs,such as miR-E (based on miR-30a) and miR-3G (based on miR-16-2) havebeen developed, and were found to exhibit stronger gene silencing inboth Pol II- and Pol III-based expression vectors in comparison toshRNAmirs, due to the enhanced processing and accumulation ofprecisely-defined guide RNAs. miR-E, which was developed by thediscovery of the conserved CNNC motif that enhances the processing ofmiRNA within the stem 3p flanking sequences, is different fromendogenous miR-30a in three aspects: the stem of miR-E has no bulge andhas the intended guide on the opposite strand; two conserved base pairsflanking the loop were mutated from CU/GG to UA/UA; and XhoI/EcoRIrestriction sites were introduced into the flanking regions for shRNAcloning (Fellmann et al. (2013) Cell Reports 5:1704-1713). miR-E wasfound to be more potent than miR-30a, but symmetric processing of boththe 3p and 5p strands of miR-30a does not favor guide strand deliveryover passenger strand delivery, which is not optimal. Additionally,cloning into miR-E using oligos longer than 100 nt is costly and timeconsuming (Watanabe et al. (2016) RNA Biology 13(1):25-33).

The amiRNA designated miR-16-2 (see, e.g., Watanabe et al. (2016) RNABiology 13(1):25-33, see FIG. 1) is a third generation (3G) amiRNAscaffold alternative; it is expressed in several tissues, is naturallyasymmetric (the mature strand is derived exclusively from the 5p or 3parm of the stem), and its stem and loop segments are small and rigid,simplifying vector cloning. miR-3G is generated by cloning the ˜175 bpfragment containing the native miR-16-2 stem and loop, and the flanking35 bps on either side of the stem, into the vector. miR-3G includesfurther modification of miR-16-2 by introducing cloning sites, such asMluI and EcoRI, into the 5p and 3p arm-flanking sequences, respectively,and fully base-pairing the guide (antisense) and passenger (sense)strand stem, with the exception of a mismatch at position 1 relative tothe guide strand. The restriction sites allow for the generation of newtargeting constructs via 88-mer duplexed DNA oligonucleotides withoutcompromising the predicted secondary structure of the miR-16-2 hairpinand flanking elements. Additionally, one of the two CNNC motifs and theGHG motif (small RNA processing enhancers) are modified in the 3pflanking sequence of miR-16-2. siRNAs targeting the gene(s) of interestare then exchanged with the first 21 nucleotides of the mature 5p guideand 3p passenger sequences. Studies determined that miR-E and miR-3Gwere equally potent. miR-3G provides an attractive RNAi system, due tothe smaller size of its expression cassette (˜175 nts vs. ˜375 formiR-E), and the simplified and cost effective single step cloning methodfor its production. As with shRNAs, bacteria can be used as vectors forthe in vivo delivery of micro-RNAs. For example, it was shown thatattenuated S. typhimurium can be used as a vector for the oral deliveryof plasmids expressing miRNA against CCL22 in mice with inflammation.Downregulation of CCL22 gene expression by this method was successfulboth in vitro and in vivo in mouse models of atopic dermatitis (Yoon etal. (2012) DNA and Cell Biology 31(3):289-296). For purposes herein amiRNA 16-2 can be used to produce miRNAs to be used in place of theshRNA. The sequences for the shRNA can be used for design of miRNAs.

DNA encoding RNAi for disrupting and/or inhibiting and/or targeting anyof selected target genes, such as any immune checkpoint described hereinor known to the skilled artisan, is inserted into a microRNA backbone,such as the microRNA backbone set forth in SEQ ID NO:249, and below. Anysuitable microRNA backbone known to the skilled artisan can be used;generally such backbones are based on a naturally-occurring microRNA andare modified for expression of the RNAi. Exemplary of such backbones isone based on miR-16-2 (SEQ ID NO:248). The sequence of the modifiedmicroRNA backbone is:

(SEQ ID NO: 249) 5′-CCGGATCAACGCCCTAGGTTTATGTTTGGATGAACTGACATACGCGTATCCGTCNNNNNNNNNNNNNNNNNNNNNGTAGTGAAATATATATTAAACNNNNNNNNNNNNNNNNNNNNNTACGGTAACGCGGAATTCGCAACTATTTTATCAATTTTTTGCGTCGAC-3′,where the N's represent complementary, generally 18-26, such as 19-24,19-22, 19-20, base pair long anti-sense and sense nucleotide sequencesthat target the gene to be silenced, and are inserted before and afterthe microRNA loop. RNAs, such as ARI-205 (SEQ ID NO:214) and ARI-206(SEQ ID NO:215) are exemplary constructs based on the microRNA backboneof SEQ ID NO:249, that encode 21 and 22 base pair homology sequences,respectively. ARI-207 (SEQ ID NO:216) and ARI-208 (SEQ ID NO:217) areexemplary constructs based on the microRNA backbone of SEQ ID NO:249,that encode 19 base pair homology sequences. Another example, is theconstruct designated ARI-201, which is microRNA construct ARI-205,wherein the N's are replaced with a sequence of nucleotides targetingmouse PD-L1. The construct designated ARI-202 represents microRNAconstruct ARI-206, where the N's are replaced with sequences targetingmouse PD-L1. The skilled person readily can construct microRNAs forinclusion in plasmids as described and exemplified herein using themiR-16-2 backbone, or other suitable backbones known to the skilledartisan.

4. Origin of Replication and Plasmid Copy Number

Plasmids are autonomously-replicating extra-chromosomal circular doublestranded DNA molecules that are maintained within bacteria by means of areplication origin. Copy number influences the plasmid stability. Highcopy number generally results in greater stability of the plasmid whenthe random partitioning occurs at cell division. A high number ofplasmids generally decreases the growth rate, thus possibly allowing forcells with few plasmids to dominate the culture, since they grow faster.The origin of replication also determines the plasmid's compatibility:its ability to replicate in conjunction with another plasmid within thesame bacterial cell. Plasmids that utilize the same replication systemcannot co-exist in the same bacterial cell. They are said to belong tothe same compatibility group. The introduction of a new origin, in theform of a second plasmid from the same compatibility group, mimics theresult of replication of the resident plasmid. Thus, any furtherreplication is prevented until after the two plasmids have beensegregated to different cells to create the correct pre-replication copynumber.

SEQ Origin Copy ID of Replication Number NO. pMB1 15-20 254 p15A 10-12255 pSC101 ~5 256 pBR322 15-20 243 ColE1 15-20 257 pPS10 15-20 258 RK2~5 259 R6K (alpha origin) 15-20 260 R6K (beta origin) 15-20 261 R6K(gamma\origin) 15-20 262 P1 (oriR) Low 263 R1 Low 264 pWSK Low 265 ColE210-15 266 pUC (pMB1) 500-700 267 F1 300-500 268

Numerous bacterial origins of replication are known to those of skill inthe art. The origin can be selected to achieve a desired copy number.Origins of replication contain sequences that are recognized asinitiation sites of plasmid replication via DNA dependent DNApolymerases (Solar et al. (1998) Microbiology And Molecular BiologyReviews 62(2):434-464). Different origins of replication provide forvarying plasmid copy levels within each cell and can range from 1 tohundreds of copies per cell. Commonly used bacterial plasmid origins ofreplication include, but are not limited to, pMB1 derived origins, whichhave very high copy derivatives, ColE1 origins, p15A, pSC101, pBR322,and others, which have low copy numbers. Such origins are well known tothose of skill in the art. The pUC 19 origin results in copy number of500-700 copies per cell. The pBR322 origin has a known copy number of15-20. These origins only vary by a single base pair. The ColE1 origincopy number is 15-20, and derivatives such as pBluescript have copynumbers ranging from 300-500. The p15A origin that is in pACYC 184, forexample, results in a copy number of approximately 10. The pSC101origins confer a copy number of approximately 5. Other low copy numbervectors from which origins can be obtained, include, for example,pWSK29, pWKS30, pWKS129 and pWKS130 (see, Wang et al. (1991) Gene100:195-199). Medium to low copy number is less than 150, or less than100. Low copy number is less than 20, 25, or 30. Those of skill in theart can identify plasmids with low or high copy number. For example, todetermine experimentally if the copy number is high or low is to performa miniprep. A high-copy plasmid should yield between 3-5 μg DNA per 1 mlLB culture; a low-copy plasmid will yield between 0.2-1 μg DNA per ml ofLB culture.

Sequences of bacterial plasmids, including identification of andsequence of the origin of replication, are well known (see, e.g.,snapgene.com/resources/plasmid_files/basic_cloning_vectors/pBR322/).

High copy plasmids are selected for heterologous expression of proteinsin vitro because the gene dosage is increased relative to chromosomalgenes and higher specific yields of protein, and for therapeuticbacteria, higher therapeutic dosages of encoded therapeutics. It isshown, herein, however, that for delivery of plasmids encoding RNAinterference (RNAi), such as by S. typhimurium, as described herein,while it would appear that a high copy plasmid would be ideally suited,therapeutically, a lower copy number is more effective.

The requirement for bacteria to maintain the high copy plasmids can be aproblem if the expressed molecule is toxic to the organism. Themetabolic requirements for maintaining these plasmids can come at a costof replicative fitness in vivo. Optimal plasmid copy number for deliveryof interfering RNAs can depend on the mechanism of attenuation of thestrain engineered to deliver the plasmid. If needed, the skilled person,in view of the disclosure herein, can select an appropriate copy numberfor a particular immunostimulatory species and strain of bacteria. It isshown herein, that low copy number can be advantageous.

5. CpG Motifs and CpG Islands

Unmethylated cytidine-phosphate-guanosine (CpG) motifs are prevalent inbacterial, but not vertebrate, genomic DNA. Pathogenic DNA and syntheticoligodeoxynucleotides (ODN) containing CpG motifs activate host defensemechanisms, leading to innate and acquired immune responses. Theunmethylated CpG motifs contain a central unmethylated CG dinucleotideplus flanking regions. In humans, four distinct classes of CpG ODN havebeen identified based on differences in structure and the nature of theimmune response they induce. K-type ODNs (also referred to as B-type)contain from 1 to 5 CpG motifs typically on a phosphorothioate backbone.D-type ODNs (also referred to as A-type) have a mixedphosphodiester/phosphorothioate backbone and have a single CpG motif,flanked by palindromic sequences that permits the formation of astem-loop structure, as well as poly G motifs at the 3′ and 5′ ends.C-type ODNs have a phosphorothioate backbone and contain multiplepalindromic CpG motifs that can form stem loop structures or dimers.P-Class CpG ODN have a phosphorothioate backbone and contain multipleCpG motifs with double palindromes that can form hairpins at theirGC-rich 3′ ends (Scheiermann and Klinman (2014) Vaccine32(48):6377-6389). For purposes herein, the CpGs are encoded in theplasmid DNA; they can be introduced as a motif, or in a gene.

Toll-like receptors (TLRs) are key receptors for sensingpathogen-associated molecular patterns (PAMPs) and activating innateimmunity against pathogens (Akira et al. (2001) Nat. Immunol.2(8):675-680). TLR9 recognizes hypomethylated CpG motifs in DNA ofprokaryotes that do not occur naturally in mammalian DNA (McKelvey etal. (2011) J. Autoimmunity 36:76-86). Recognition of CpG motifs uponphagocytosis of pathogens into endosomes in immune cell subsets inducesIRF7-dependent type I interferon signaling and activates innate andadaptive immunity.

Immunostimulatory bacteria, such as Salmonella species, such as S.typhimurium, strains carrying plasmids containing CpG islands, areprovided herein. These bacteria can activate TLR9 and induce type IIFN-mediated innate and adaptive immunity. As exemplified herein,bacterial plasmids that contain hypomethylated CpG islands can elicitinnate and adaptive anti-tumor immune responses that, in combinationwith RNAi encoded in the plasmid, such as RNAi that targets immunecheckpoints, such as the shRNA or miRNA that targets TREX1, and hence,TREX1-mediated STING pathway activation, can have synergistic orenhanced anti-tumor activity. For example, the asd gene (SEQ ID NO:48)encodes a high frequency of hypomethylated CpG islands. CpG motifs canbe included in combination with any of the RNAi described or apparentfrom the description herein in the immunostimulatory bacteria, andthereby enhance or improve anti-tumor immune responses in a treatedsubject.

Immunostimulatory CpGs can be included in the plasmids, by including anucleic acid, typically from a bacterial gene, that encodes a geneproduct, and also by adding a nucleic acid that encodes CpG motifs. Theplasmids herein can include CpG motifs. Exemplary CpG motifs are known(see, e.g., U.S. Pat. Nos. 8,232,259, 8,426,375 and 8,241,844). Theseinclude, for example, synthetic immunostimulatory oligonucleotides,between 10 and 100, 10 and 20, 10 and 30, 10 and 40, 10 and 50, 10 and75, base pairs long, with the general formula:

(CpG)_(n), where n is the number of repeats.

Generally, at least one or two repeats are used; non-CG bases can beinterspersed. Those of skill in the art are very familiar with thegeneral use of CpG motifs for inducing an immune response by modulatingTLRs, particularly TLR9.

6. Plasmid Maintenance/Selection Components

The maintenance of plasmids in laboratory settings is usually ensured bythe inclusion of an antibiotic resistance gene on the plasmid and use ofantibiotics in the growth media. As described above, the use of an asddeletion mutant complimented with a functional asd gene on the plasmidallows for plasmid selection in vitro without the use of antibiotics,and allows for plasmid maintenance in vivo. The asd gene complementationsystem provides for such selection/maintenance (Galán et al. (1990) Gene28:29-35). The use of the asd gene complementation system to maintainplasmids in the tumor microenvironment increases the potency of S.typhimurium and other immunostimulatory bacterial strains, engineered todeliver plasmids encoding therapeutic products such as immunostimulatoryproteins, antibodies, antibody fragments, or interfering RNAs.

7. RNA Polymerase Promoters

Plasmids provided herein are designed to encode interfering RNAstargeting immune checkpoints and other targets as described above. TheRNA expression cassette contains a promoter for transcription in humancells such as an H1 promoter or a U6 promoter, or a CMV promoter. U6 andH1 are RNA polymerase III (RNAP III) promoters, which are for productionand processing of small RNAs. The CMV promoter is recognized by RNApolymerase II, and is more amenable for expression of long RNA stretchesthan is RNAP III. The promoter precedes the interfering RNA, such as anshRNA, siRNA or miRNA, as described above.

In eukaryotic cells, DNA is transcribed by three types of RNApolymerases; RNA Pol I, II and III. RNA Pol I transcribes only ribosomalRNA (rRNA) genes, RNA Pol II transcribes DNA into mRNA and small nuclearRNAs (snRNAs), and RNA Pol III transcribes DNA into ribosomal 5S rRNA(type I), transfer RNA (tRNA) (type II) and other small RNAs such as U6snRNAs (type III). shRNAs are typically transcribed in vivo under thecontrol of eukaryotic type III RNA Pol III promoters, such as the humanU6 promoter, which transcribes the U6 snRNA component of thespliceosome, and the H1 human promoter, which transcribes the RNAcomponent of RNase P. U6 and H1 promoters are more suitable than otherPol III or Pol II promoters because they are structurally simple, with awell-defined transcription start-site, and naturally drive thetranscription of small RNAs. U6 and H1 promoters do not carry thesequences necessary for transcribing anything downstream from thetranscription start site (Makinen et al. (2006) J. Gene Med. 8:433-441).They are thus the most straightforward promoters for use in shRNAexpression.

The use of other promoters such as type II pol III tRNA promoters, whilesuccessful in expressing shRNAs, results in longer dsRNA transcripts,which can induce an interferon response. RNA pol II promoters, such asthe human cytomegalovirus (CMV) promoter also can be used (U.S. Pat.Nos. 8,202,846 and 8,383,599), but are more often utilized forexpression of long RNA stretches. Studies have shown that the additionof the enhancer from the CMV promoter near the U6 promoter can increaseits activity, increasing shRNA synthesis and improving gene silencing(Xia et al. (2003) Nucleic Acids Res. 31(17):e100; Nie et al. (2010)Genomics Proteomics Bioinformatics 8(3):170-179). RNA pol II promotersare typically avoided in shRNA transcription due to the generation ofcytoplasmic DNA, which leads to a pro-inflammatory interferon response.In this case, a cytoplasmic DNA mediated interferon response in S.typhimurium-infected tumor cells has anti-tumor benefit, especially inthe context of TREX1 inhibition as provided herein. Prokaryoticpromoters, including T7, pBAD and pepT promoters can be utilized whentranscription occurs in a bacterial cell (Guo et al. (2011) Gene therapy18:95-105; U.S. Patent Publication Nos. 2012/0009153, 2016/0369282;International Application Publication Nos. WO 2015/032165, WO2016/025582).

RNA pol III promoters generally are used for constitutive shRNAexpression. For inducible expression, RNA pol II promoters are used.Examples include the pBAD promoter, which is inducible by L-arabinose;tetracycline-inducible promoters such as TRE-tight, IPT, TRE-CMV, Tet-ONand Tet-OFF; retroviral LTR; IPTG-inducible promoters such as LacI,Lac-O responsive promoters; LoxP-stop-LoxP system promoters (U.S. Pat.No. 8,426,675; International Application Publication No. WO2016/025582); and pepT, which is a hypoxia-induced promoter. (Yu et al.(2012) Scientific Reports 2:436). These promoters are well known.Exemplary of these promoters are human U6 (SEQ ID NO:73) and human H1(SEQ ID NO:74).

SEQ ID NO. Name Sequence 73 human U6                                             aa ggtcgggcag gaagagggccRNA pol721 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattaIII781 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaatpromoter841 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta901 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaact961 ag 74 human H1                                                 atatttgca tgtcgctatgRNA pol721 tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct tataagttctIII 781 gtatgagacc actccctagg promoter

Tissue specific promoters include TRP2 promoter for melanoma cells andmelanocytes; MMTV promoter or WAP promoter for breast and breast cancercells, Villin promoter or FABP promoter for intestinal cells, RIPpromoter for pancreatic beta cells, Keratin promoter for keratinocytes,Probasin promoter for prostatic epithelium, Nestin promoter or GFAPpromoter for CNS cells/cancers, Tyrosine Hydroxylase S 100 promoter orneurofilament promoter for neurons, Clara cell secretory proteinpromoter for lung cancer, and Alpha myosin promoter in cardiac cells(U.S. Pat. No. 8,426,675).

8. DNA Nuclear Targeting Sequences

DNA nuclear targeting sequences (DTS) such as the SV40 DTS mediate thetranslocation of DNA sequences through the nuclear pore complex. Themechanism of this transport is reported to be dependent on the bindingof DNA binding proteins that contain nuclear localization sequences. Theinclusion of a DTS on a plasmid to increase nuclear transport andexpression has been demonstrated (Dean, D. A. et al. (1999) Exp. CellRes. 253(2):713-722), and has been used to increase gene expression fromplasmids delivered by S. typhimurium (Kong et al. (2012) Proc. Natl.Acad. Sci. U.S.A. 109(47):19414-19419).

Rho-independent or class I transcriptional terminators such as the T1terminator of the rrnB gene of E. coli contain sequences of DNA thatform secondary structures that cause dissociation of the transcriptionelongation complex. Transcriptional terminators shall be included in theplasmid in order to prevent expression of interfering RNAs by the S.typhimurium transcriptional machinery. This ensures that expression ofthe encoded interfering RNA, such as shRNA, micro-RNA and siRNA, isconfined to the host cell transcriptional machinery.

Plasmids used for transformation of Salmonella, such as S. typhimurium,as a cancer therapy described herein, contain all or some of thefollowing attributes: 1) a CpG island, 2) a bacterial origin ofreplication, 3) an asd gene selectable marker for plasmid maintenance,4) one or more human interfering RNA expression cassettes, 5) DNAnuclear targeting sequence, and 6) transcriptional terminators.

9. CRISPR

An immunostimulatory bacterium, encoding a CRISPR cassette, can be usedto infect human immune, myeloid, or hematopoietic cells in order tosite-specifically knockout a target gene of interest. The strain usedcan be asd⁻ and can contain a plasmid that lacks the complementary asdcassette and contains a kan cassette. In order to grow the strain invitro in liquid media, DAP is added to complement the asd⁻ geneticdeficiency. After infection of human cells, the strain can no longerreplicate, and the CRISPR cassette-encoded plasmid is delivered. Thestrain also can be hilA⁻ or can lack one or more parts of SPI-1, or lackflagellin, or any combination thereof, which reduces or preventspyroptosis (inflammatory-mediated cell death) of phagocytic cells.

G. Tumor-Targeting Immunostimulatory Bacteria Contain RNAi AgainstExemplary Immune Target Genes to Stimulate Anti-Tumor Immunity

RNAi against any immune target can be encoded in the plasmids. Theseinclude, but are not limited to, any discussed in the disclosure herein,and any known to those of skill in the art. The following discussiondescribes exemplary targets. The plasmids can contain any RNAi againstsuch targets, including, but not limited to, shRNA, siRNA and microRNA.

1. TREX1

In certain embodiments provided herein, the immunostimulatory bacteriaencode inhibitory RNA, such as shRNA, that inhibit or disrupt orsuppress TREX1 expression. The enzyme product encoded by TREX1, locatedupstream from cGAS, is a mediator of the type I interferon pathway.TREX1 encodes the major 3′ DNA exonuclease in mammalian cells (alsocalled DNase III). Human TREX1 proteins are as catalytically efficientas bacterial exonucleases (Mazur and Perrino (2001) J. Biol. Chem.276:17022-17029). Immunostimulatory bacterium that inhibit TREX1expression by processes other than RNA silencing also are contemplatedherein.

For the immunostimulatory bacteria provided herein, such as those thatexpress shRNA against TREX1, loss of TREX1 activity and subsequentactivation of cGAS/STING-induced vascular disruption enhances tumorcolonization of S. typhimurium. The TREX1 gene encodes a protein that is314 amino acids long (Mazur et al. (2001) J. Biol. Chem276:17022-17029), exists as a homodimer, and lacks endonucleaseactivity. TREX1 is among several proteins involved in the repair of DNAthat is damaged by exogenous genotoxic stress, including UV irradiationand DNA-damaging compounds. TREX1 can function as an editing exonucleasefor DNA pol β by excising mispaired nucleotides from the 3′ end (Mazuret al. (2001) J. Biol. Chem 276:17022-17029). ssDNA is degraded 3-4times more efficiently than dsDNA (Lindahl et al. (2009) Biochem. Soc.Trans. 37 (Pt 3), 535-538). Mutations in residues D18 and D200,frequently associated with autoimmune diseases, disable TREX1 enzymefrom degrading dsDNA and reduces its ability to degrade ssDNA. TREX1enzyme translocates from the endoplasmic reticulum to the nucleusfollowing DNA damage, indicating its involvement in the replication ofdamaged DNA. Promoter activation and upregulation of TREX1 has beenobserved as a result of UVC exposure in mouse fibroblasts, and TREX1null mouse cells have demonstrated hypersensitivity to UVC light(Tomicic et al. (2013) Bioch. Biophys. Acta 1833:1832-1843).

Mutations resulting in loss of TREX1 have been identified in patientswith the inherited rare disease, Aicardi-Goutieres syndrome (AGS), whichhas phenotypic overlap with the autoimmune diseases systemic lupuserythematosus (SLE) and chilblain lupus (Aicardi and Goutieres, (2000)Neuropediatrics 31(3): 113). Mutations in TREX1 also are associated withretinal vasculopathy with cerebral leukodystrophy. TREX1-mediatedautoimmune diseases are associated with the cell's inability to preventautoimmunity via the degradation of ssDNA and dsDNA that accumulates inthe cytoplasm. TREX1 null mice suffer from inflammatory myocarditis,resulting in circulatory failure, which is caused by chronic cytokineproduction (Morita et al. (2004) Mol. Cell Biol. 24(15):6719-6727; Yanget al. (2007) Cell 131(5):873-886; Tomicic et al. (2013) Bioch. Biophys.Acta 1833(8):1832-1843). Hence, TREX1 deficiency induces innate immunityfollowing the cytoplasmic accumulation of DNA, resulting in aninflammatory response (Wang et al. (2009) DNA Repair(Amst) 8:1179-1189).The source of the DNA that accumulates in the cytosol of TREX1-deficientcells was found to be in part derived from endogenous retroelements thatescape from the damaged nucleus, as TREX1 is known to metabolizereverse-transcribed (RT) DNA (Stetson et al. (2008) Cell134(4):587-598). In HIV infection, HIV RT DNA accumulates in the cytosolof infected T cells and macrophages, and would normally triggercGAS/STING activation of antiviral immunity. TREX1 digests this viralDNA and permits HIV immune escape (Yan et al. (2010) Nat. Immunol.11(11): 1005-1013). Thus, TREX1 acts as a negative regulator of STING,and can be exploited to evade detection by several retroviruses, such asmurine leukemia virus (MLV), simian immunodeficiency virus (SIV), andmany others (Hasan et al. (2014) Front. Microbiol. 5:193).

Like STING, TREX1 is expressed in most mammalian cell types, with thekey producers of cytokines in TREX1 null mice originating frommacrophages and dendritic cells (Ahn et al. (2014) J. Immunol.193(9):4634-4642). Data indicate that TREX1 is responsible for degradingself-DNA that can leak from a damaged nucleus into the cytosol, where itwould otherwise bind and activate cGAS and lead to autoimmunity (Barber(2015) Nat. Rev. Immunol. 15(12):760-770). In support of this, TREX1null mice and TREX1-deficient cells that also lack cGAS are completelyprotected from type I interferon activation and lethal autoimmunity(Ablasser et al. (2014) J. Immunol. 192(12):5993-5997; Gray et al.(2015) J. Immunol. 195(5):1939-1943). In a negative feedback loop, typeI interferon and type II IFNγ can also induce TREX1, and TREX1 thusserves to limit aberrant autoimmune activation (Tomicic et al. (2013)Bioch. Biophys. Acta 1833:1832-1843).

Lymphocytes derived from an Aicardi-Goutieres syndrome patient,containing mutated TREX1, were found to inhibit angiogenesis and thegrowth of neuroblastoma cells, the effect being enhanced by the presenceof IFN-α (Pulliero et al. (2012) Oncology Reports 27:1689-1694). The useof microRNA-103 also has been shown to inhibit the expression of TREX1,disrupting DNA repair and angiogenesis, and resulting in decreased tumorgrowth in vivo (see, U.S. Patent Publication No. 2014/0127284, Chereshet al.).

TREX1 is a negative regulator of macrophage activation andpro-inflammatory function. TREX1 null macrophages were found to exhibitincreased TNF-α and IFN-α production, higher levels of CD86, andincreased antigen presentation to T cells, as well as impaired apoptoticT cell clearance (Pereira-Lopes et al. (2013) J. Immunol.191:6128-6135). The inability to adequately digest apoptotic DNA inTREX1 null macrophages generates high amounts of aberrant cytosolic DNA,which binds to cGAS and activates the STING pathway to produce higherlevels of type I interferon (Ahn et al. (2014) J. Immunol.193:4634-4642). Not all cell types are sensitive to theimmunostimulatory effects of Trex1 knockdown, however. In a study ofindividual cell types, dendritic cells, macrophages, fibroblasts andkeratinocytes were found to produce type I IFN upon TREX1 knockdown,while B cells, cardiomyocytes, neurons and astrocytes did not (Peschkeet al. (2016) J. Immunol. 197:2157-2166). Thus, inhibiting the functionof TREX1 in phagocytic cells that have engulfed S. typhimurium wouldenhance their pro-inflammatory activity, while driving an accumulationof cytosolic DNA from phagocytosed tumor cells that can then activatethe cGAS/STING pathway. The use of microRNA-103 has inhibits theexpression of TREX1, disrupting DNA repair and angiogenesis, andresulting in decreased tumor growth in vivo (see, U.S. Publication No.2014/0127284, Cheresh et al.).

Studies have found that the expression of cGAS and/or STING is inhibitedin over a third of colorectal cancers, while STING expression is lost inmany primary and metastatic melanomas and HPV⁺ cancers. STING signalingremains intact in all tumor-resident APCs that continuously sample theantigenic milieu of the TME, including Batf3-lineage CD103/CD8c⁺ DCsthat cross-present tumor antigens to CD8⁺ T cells, and these APCs willalso readily phagocytose S. typhimurium or be activated by type I IFNfrom neighboring macrophages that have phagocytosed S. typhimuriumcontaining TREX1 gene knockdown.

Inactivation of TREX1 enhances an immune response by permittingcytosolic accumulation of dsDNA to bind to the enzyme cyclic GMP-AMP(cGAMP) synthase (cGAS), a cytosolic DNA sensor that triggers theproduction of type I interferons and other cytokines through activationof the STING signaling pathway (Sun et al. (2013) Science339(6121):786-791; Wu et al. (2013) Science 339(6121):826-830).Activation of the STING pathway has been shown to induce potent innateand adaptive antitumor immunity (Corrales et al. (2015) Cell Reports11:1018-1030).

Hence, embodiments of the immunostimulatory bacterial strains, asprovided herein, are administered to inhibit TREX1 in tumor-residentAPCs and induce cGAS/STING activation, thereby activating these DCs tocross-present host tumor antigens to CD8⁺ T cells and induce local andsystemic tumor regression and durable anti-tumor immunity (Corrales etal. (2015) Cell Reports 11:1018-1030; Zitvogel et al. (2015) Nat. Rev.Mol. Cell. Biol. 16:393-405).

Immunostimulatory bacteria provided herein express RNAi against TREX1,and loss of TREX1 and subsequent activation of cGAS/STING-inducedvascular disruption enhance tumor colonization of S. typhimurium.

2. PD-L1

Programmed cell death protein 1 (PD-1) is an immune-inhibitory receptorthat is involved in the negative regulation of immune responses. Itscognate ligand, programmed death-ligand 1 (PD-L1), is expressed on APCs,and upon binding to PD-1 on T cells, leads to loss of CD8⁺ T celleffector function, inducing T cell tolerance. The expression of PD-L1 isoften associated with tumor aggressiveness and reduced survival incertain human cancers (Gao et al. (2009) Clin. Cancer Res.15(3):971-979).

Antibodies designed to block immune checkpoints, such as anti-PD-1 (forexample, pembrolizumab, nivolumab) and anti-PD-L1 (for example,atezolizumab, avelumab, durvalumab) antibodies have had durable successin preventing T cell anergy and breaking immune tolerance. Only afraction of treated patients exhibit clinical benefit, and those that dooften present with autoimmune-related toxicities (Ribas (2015) N. Engl.J. Med. 373(16):1490-1492; Topalian et al. (2012) N. Engl. J. Med.366(26):2443-54). Besides acquiring toxicity, PD-1/PD-L1 therapy oftenleads to resistance, and the concomitant use of anti-CTLA-4 antibodies(for example, ipilimumab) has shown limited success in clinical trialswith significantly additive toxicity. To limit the toxicity and enhancethe potency of PD-L1 blockade, an immunostimulatory bacteria with anshRNA to PD-L1, as provided herein, will synergize with TLR activationof immune cells to both activate and potentiate anti-tumor immunity.

3. VISTA

Other non-redundant checkpoints in immune activation can synergize withPD-1/PD-L1 and CTLA-4, such as V-domain immunoglobulin (Ig) suppressorof T cell activation (VISTA). VISTA is expressed primarily on APCs,particularly on tumor-infiltrating myeloid cells and myeloid-derivedsuppressor cells (MDSC), and to a lesser extent on regulatory T cells(CD4⁺ Foxp3⁺ Tregs) (Wang et al. (2011) J. Exp. Med. 208(3):577-592).Similar to PD-L1, VISTA upregulation directly suppresses T cellproliferation and cytotoxic function (Liu et al. (2015) Proc. Natl.Acad. Sci. U.S.A. 112(21):6682-6687). Monoclonal antibody targeting ofVISTA was shown to remodel the tumor microenvironment in mice,increasing APC activation and enhancing anti-tumor immunity (LeMercieret al. (2014) Cancer Res. 74(7): 1933-1944). Clinically, VISTAexpression was shown to be upregulated on tumor-resident macrophagesfollowing treatment with anti-CTLA-4 therapy in prostate cancer,demonstrating compensatory regulation of immune checkpoints (Gao et al.(2017) Nat. Med. 23(5):551-555). The majority of VISTA expression ispurported to be located in the intracellular compartment of myeloidcells, rather than on the surface, which can limit the effectiveness ofthe monoclonal antibody approach (Deng et al. (2016) J. Immunother.Cancer 4:86). The ability to inhibit VISTA from within the APC using atumor-targeting bacteria containing shRNA to VISTA, as provided herein,will more efficiently and completely inhibit the T cell-suppressingfunction of VISTA, leading to activation of T cell-mediated anti-tumorimmunity and tumor regression.

4. SIRPα

One mechanism by which tumor cells evade removal is to prevent theirphagocytosis by innate immune cells. Phagocytosis is inhibited bysurface expression of CD47, which is widely expressed on hematopoieticand non-hematopoietic cells (Liu et al. (2015) PLoS ONE 10(9):e0137345).Upon CD47 binding its receptor, signal regulatory protein alpha (SIRPα),an inhibitory signal for phagocytosis, is initiated. SIRPα is abundantlyexpressed on phagocytic cells, including macrophages, granulocytes andDCs. As such, the protein-protein interaction between CD47 and SIRPαrepresents another class of immune checkpoints unique to APCs, andtumor-resident macrophages in particular. The effectiveness of CD47 inpreventing phagocytosis is evidenced by the fact that it is oftenupregulated in a wide variety of tumors, which allow them to avoid beingphagocytosed by APCs in the tumor microenvironment (Liu et al. (2015)Nat. Med. 21(10):1209-1215). Several methods to block the CD47/SIRPαinteraction have been examined, including the development of anti-CD47or anti-SIRPα antibodies or antibody fragments, the use of smallpeptides that bind either protein, or the knockdown of CD47 expression(U.S. Patent Publication Nos. 2013/0142786, 2014/0242095; InternationalApplication Publication No. WO 2015/191861; McCracken et al. (2015)Clin. Cancer Res. 21(16):3597-3601). To this end, several monoclonalantibodies that directly target SIRPα are in clinical development,either alone or in combination with tumor-targeting antibodies (e.g.,Rituximab, Daratumumab, Alemtuzumab, Cetuximab) that can enhancephagocytosis of antibody-opsonized tumor cells, in a process known asantibody-dependent cellular phagocytosis (ADCP) (McCracken et al. (2015)Clin. Cancer Res. 21(16):3597-3601; Yanagita et al. (2017) JCI Insight2(1):e89140).

The CD47/SIRPα interaction also serves to preserve the longevity of redblood cells by preventing their phagocytic elimination (Murata et al.(2014) J. Biochem. 155(6):335-344). Thus, systemically administeredtherapies such as anti-CD47 antibodies that broadly disrupt thisinteraction have resulted in anemia toxicities (Huang et al. (2017) J.Thorac. Dis. 9(2):E168-E174). Systemic SIRPα-based therapies also riskadverse events, such as organ damage by creating systemichyperphagocytic self-eating macrophages. Using a tumor-targetingimmunostimulatory bacteria containing an shRNA to SIRPα, such asprovided herein, will localize the CD47/SIRPα disruption to the tumormicroenvironment and eliminate these adverse events. Further, inhibitionof SIRPα in the context of bacterial activation of TLR-mediatedpro-inflammatory signaling pathways will potently activate thesemacrophages to become hyperphagocytic towards neighboring tumor cells(Bian et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113(37): E5434-E5443).

5. β-catenin

Immune checkpoint pathways exemplify the multiple layers of regulationthat exist to prevent immune hyper-activation and autoimmunity, and thedifficulties in subverting these pathways to promote anti-tumorimmunity. One mechanism by which tumors have evolved to be refractory tocheckpoint therapies is through their lack of T cell and dendritic cell(DC) infiltration, described as non-T-cell-inflamed, or “cold tumors”(Sharma et al. (2017) Cell 9; 168(4):707-723). Several tumor-intrinsicmechanisms have been identified that lead to the exclusion of anti-tumorT cells and resistance to immunotherapy. In melanoma, in particular,molecular profiling of checkpoint therapy-refractory tumors revealed asignature of elevated β-catenin and its downstream target genes,correlating with a lack of tumor-infiltrating lymphocytes (Gajewski etal. (2011) Curr. Opin. Immunol. 23(2):286-292).

CTNNB1 is an oncogene that encodes β-catenin, and can induce theexpression of the genes c-Myc and cyclin D1, resulting in tumorproliferation. Mutations in CTNNB1 are associated with certain cancers.Gene silencing of CTNNB1/β-catenin using S. typhimurium shRNA vectorscan be used in the treatment of cancer (Guo et al. (2011) Gene therapy18:95-105; U.S. Patent Publication Nos. 2012/0009153, 2016/0369282;International Patent Publication No. WO 2015/032165). For example, shRNAsilencing of CTNNB1, using S. typhimurium strain SL7207 as a deliveryvector, reduced tumor proliferation and growth in SW480 xenograft mice,when compared to control cells, and reduced expression of c-Myc andcyclin D1(Guo et al. (2011) Gene therapy 18:95-105). Silencing of CTNNB1for the treatment of hepatoblastoma also can be achieved using miRNA,with or without antibody therapeutics against the immune checkpointsPD-land PD-L1 (International Application Publication No. WO2017/005773). The use of siRNA or shRNA targeting CTNNB1, delivered viaalternative vectors, such as liposomes, for the treatment ofCTNNB1-related cancers, including adenocarcinomas and squamous cellcarcinomas, also can be affected (U.S. Patent Publication Nos.2009/0111762, 2012/0294929).

Elevated β-catenin signaling directly inhibits the chemokine CCL4 fromrecruiting Batf3-lineage CD103/CD8α⁺ DCs, thereby preventing them frompriming tumor antigen-specific CD8⁺ T cells (Spranger et al. (2015)Nature 523(7559):231-235). β-catenin is the major downstream mediator ofthe WNT signaling pathway, a key embryonic developmental pathway that isalso critical for adult tissue regeneration, homeostasis andhematopoiesis (Clevers et al. (2012) Cell 149(6):1192-1205). ExcessiveWNT/β-catenin signaling has been implicated in a variety of cancers (Taiet al. (2015) Oncologist 20(10):1189-1198). Accordingly, severalstrategies to target WNT/β-catenin signaling have been pursued, butsuccess has been hampered by a lack of specificity to the tumormicroenvironment, resulting in off-target toxicities to intestinal stemcells, bone turnover and hematopoiesis (Kahn (2014) Nat. Rev. Drug Dis.13(7):513-532). The immunostimulatory bacteria provided herein overcomethese problems.

For example, an advantage of using an immunostimulatory bacteria withshRNA to β-catenin as provided herein, is enhancing chemokine-mediatedinfiltration of T cell-priming DCs and the conversion of a cold tumor toa T-cell-inflamed tumor microenvironment, without the systemictoxicities of existing therapeutic modalities. Further, bacterialactivation of TLR innate immune signaling pathways synergize withβ-catenin inhibition to further promote immune activation and anti-tumorimmunity.

6. TGF-β

Transforming growth factor beta (TGF-β) is a pleiotropic cytokine withnumerous roles in embryogenesis, wound healing, angiogenesis and immuneregulation. It exists in three isoforms in mammalian cells, TGF-β1,TGF-β2 and, TGF-β3; TGF-β1 is the most predominant in immune cells(Esebanmen et al. (2017) Immunol Res. 65:987-994). TGF-β's role as animmunosuppressant is arguably its most dominant function. Its activationfrom a latent form in the tumor microenvironment, in particular, hasprofound immunosuppressive effects on DCs and their ability to tolerizeantigen-specific T cells. TGF-β can also directly convert Th1 CD4⁺ Tcells to immunosuppressive Tregs, furthering promoting tumor tolerance(Travis et al. (2014) Annu. Rev. Immunol. 32:51-82). Based on itstumor-specific immunosuppressive functions, and irrespective of itsknown cancer cell growth and metastasis-promoting properties, inhibitionof TGF-β is a cancer therapy target. High TGF-β signaling has beendemonstrated in several human tumor types, including CRC, HCC, PDAC andNSCLC (Colak et al. (2017) Trends in Cancer 3:1). Systemic inhibition ofTGF-β can lead to unacceptable autoimmune toxicities, and its inhibitionshould be localized to the tumor microenvironment. As such, atumor-targeting immunostimulatory bacteria with RNAi, such as shRNA, toTGF-β, provided herein, or an shRNA to TGF-βRII, breaks tumor immunetolerance and stimulates anti-tumor immunity.

7. VEGF

Angiogenesis, or the development of new blood vessels, is an essentialstep for any tumor microenvironment to become established. Vascularendothelial growth factor (VEGF) is the critical mitogen for endothelialproliferation and angiogenesis, and inhibition of VEGF in the tumormicroenvironment markedly decreases tumor vascularity, thereby starvingthe tumor of its blood supply (Kim et al. (1993) Nature362(6423):841-4). This early research led to the development of themonoclonal antibody inhibitor of VEGF, bevacizumab (Avastin; Genentech),which in combination with chemotherapy, has become the standard of carefor metastatic CRC. Systemic administration of bevacizumab alsodemonstrated significant toxicities, including multiple fatalities in aPhase II trial of NSCLC, largely due to hemorrhaging. As such, severalnext generation anti-angiogenics have been evaluated, such as theanti-VEGF receptor 2 antibody ramucirumab (Cyramza, Imclone) and theanti-angiogenic tyrosine kinase inhibitor axitinib (Inlyta, Pfizer), yetnone have been able to overcome systemic toxicity or markedly improveprogression-free survival (Alshangiti et al. (2018) Curr. Oncol.25(Suppl 1):S45-S58). While the anti-tumor activity of anti-VEGF therapyhas shown some promise, systemic toxicity is clearly limiting. As such,a therapy that targets only the tumor microenvironment, such as animmunostimulatory tumor-targeting bacteria with shRNA to VEGF, providedherein, delivers local anti-angiogenic therapy while preventing systemictoxicity. This therapeutic modality has the additional advantage ofbeing taken up into myeloid cells, which predominantly produce VEGF inthe tumor microenvironment, where it will have maximum impact on tumorprogression (Osterberg et al. (2016) Neuro-Oncology. 18(7):939-949).

8. Additional Exemplary Checkpoint Targets

Exemplary checkpoint targets for which RNAi, such as micro-RNA andshRNA, can be prepared or are exemplified herein include, but are notlimited to:

Checkpoint target CTLA-4 PD-L1 (B7-H1) PD-L2 PD-1, PD-2 IDO1 IDO2 SIRPalpha, CD47 VISTA (B7-H5) LIGHT HVEM CD28 LAG3, TIM3, TIGIT Galectin-9CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, B7-H3, CD137, ICOS,GITR, B7-H4. B7-H6 CD137, CD27, CD40/CD40L, CD48, CD70, CD80, CD86,CD137 (4-1BB), CD200, CD272 (BTLA), CD160 A2a receptor, A2b receptor,HHLA2, ILT-2, ILT-4, gp49B, PIR-B OX40/OX-40L, BTLA, ICOS, HLA-G,ILT-2/4 KIR, GITR, TIM1, TIM4Other exemplary targets include, but are not limited to:

Target CTNNB1 (beta-catenin) STAT3 BCL-2 MDR1 Arginase1 iNOS TGF-β IL-10pGE2 VEGF KSP HER2 KRAS TAK1 PLK1 K-Ras (Ras) Stablin-1/CLEVER-1 RNaseH2 DNase II

H. Pharmaceutical Production, Compositions, and Formulations

Provided herein are methods for manufacturing, pharmaceuticalcompositions and formulations containing any of the immunostimulatorybacteria provided herein and pharmaceutically acceptable excipients oradditives. The pharmaceutical compositions can be used in treatment ofdiseases, such as hyperproliferative diseases or condition, such as atumor or cancer. The immunostimulatory bacteria can be administered in asingle agent therapy, or can be administered in a combination therapywith a further agent or treatment. The compositions can be formulatedfor single dosage administration or for multiple dosage administration.The agents can be formulated for direct administration. The compositionscan be provided as a liquid or dried formulation.

1. Manufacturing

a. Cell Bank Manufacturing

As the active ingredient of the immunotherapeutic described herein iscomposed of engineered self-replicating bacteria, the selectedcomposition will be expanded into a series of cell banks that will bemaintained for long-term storage and as the starting material formanufacturing of drug substance. Cell banks are produced under currentgood manufacturing practices (cGMP) in an appropriate manufacturingfacility per the Code of Federal Regulations (CFR) 21 part 211 or otherrelevant regulatory authority. As the active agent of theimmunotherapeutic is a live bacterium, the products described hereinare, by definition, non-sterile and cannot be terminally sterilized.Care must be taken to ensure that aseptic procedures are used throughoutthe manufacturing process to prevent contamination. As such, all rawmaterials and solutions must be sterilized prior to use in themanufacturing process.

A master cell bank (MCB) is produced by sequential serial single colonyisolation of the selected bacterial strain to ensure no contaminants arepresent in the starting material. A sterile culture vessel containingsterile media (can be complex media e.g., LB or MSBB or defined mediae.g., M9 supplemented with appropriate nutrients) is inoculated with asingle well-isolated bacterial colony and the bacteria are allowed toreplicate e.g., by incubation at 37° C. with shaking. The bacteria arethen prepared for cryopreservation by suspension in a solutioncontaining a cryoprotective agent or agents.

Examples of cryoprotective agents include: proteins such as human orbovine serum albumin, gelatin, immunoglobulins; carbohydrates includingmonosaccharides (galactose, D-mannose, sorbose, etc.) and theirnon-reducing derivatives (e.g., methylglucoside), disaccharides(trehalose, sucrose, etc.), cyclodextrins, and polysaccharides(raffinose, maltodextrins, dextrans, etc.); amino-acids (glutamate,glycine, alanine, arginine or histidine, tryptophan, tyrosine, leucine,phenylalanine, etc.); methylamines such as betaine; polyols such astrihydric or higher sugar alcohols, e.g., glycerin, erythritol,glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol;polyethylene glycol; surfactants e.g., pluronic; or organo-sulfurcompounds such as dimethyl sulfoxide (DMSO), and combinations thereof.Cryopreservation solutions can include one or more cryoprotective agentsin a solution that can also contain salts (e.g., sodium chloride,potassium chloride, magnesium sulfate, and or buffering agents such assodium phosphate, tris(hydroxymethyl)aminomethane (TRIS),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and othersuch buffering agents known to those of skill.

Suspension of the bacteria in cryopropreservation solution can beachieved either by addition of a concentrated cryoprotective agent oragents to the culture material to achieve a final concentration thatpreserves viability of the bacteria during the freezing and thawingprocess (e.g., 0.5% to 20% final concentration of glycerol), or byharvesting the bacteria (e.g., by centrifugation) and suspending in acryopreservative solution containing the appropriate final concentrationof cryoprotective agent(s). The suspension of bacteria incryopreservation solution is then filled into appropriate sterile vials(plastic or glass) with a container closure system that is capable ofmaintaining closure integrity under frozen conditions (e.g., butylstoppers and crimp seals). The vials of master cell bank are then frozen(either slowly by means of a controlled rate freezer, or quickly bymeans of placing directly into a freezer). The MCB is then stored frozenat a temperature that preserves long-term viability (e.g., at or below−60° C.). Thawed master cell bank material is thoroughly characterizedto ensure identity, purity, and activity per regulation by theappropriate authorities.

Working cell banks (WCBs) are produced much the same way as the mastercell bank, but the starting material is derived from the MCB. MCBmaterial can be directly transferred into a fermentation vesselcontaining sterile media and expanded as above. The bacteria are thensuspended in a cryopreservation solution, filled into containers,sealed, and frozen at or below −20° C. Multiple WCBs can be producedfrom MCB material, and WCB material can be used to make additional cellbanks (e.g., a manufacturer's working cell bank MWCB). WCBs are storedfrozen and characterized to ensure identity, purity, and activity. WCBmaterial is typically the starting material used in production of thedrug substance of biologics such as engineered bacteria.

b. Drug Substance Manufacturing

Drug substance is manufactured using aseptic processes under cGMP asdescribed above. Working cell bank material is typically used asstarting material for manufacturing of drug substance under cGMP,however other cell banks can be used (e.g., MCB or MWCB). Asepticprocessing is used for production of all cell therapies includingbacterial cell-based therapies. The bacteria from the cell bank areexpanded by fermentation, this can be achieved by production of apre-culture (e.g., in a shake flask) or by direct inoculation of afermenter. Fermentation is accomplished in a sterile bioreactor or flaskthat can be single-use disposable or re-usable. Bacteria are harvestedby concentration (e.g., by centrifugation, continuous centrifugation, ortangential flow filtration). Concentrated bacteria are purified frommedia components and bacterial metabolites by exchange of the media withbuffer (e.g., by diafiltration). The bulk drug product is formulated andpreserved as an intermediate (e.g., by freezing or drying) or isprocessed directly into a drug product. Drug substance is tested foridentity, strength, purity, potency, and quality.

c. Drug Product Manufacturing

Drug product is defined as the final formulation of the active substancecontained in its final container. Drug product is manufactured usingaseptic processes under cGMP. Drug product is produced from drugsubstance. Drug substance is thawed or reconstituted if necessary, thenformulated at the appropriate target strength. Because the activecomponent of the drug product is live, engineered bacteria, the strengthis determined by the number of CFUs contained within the suspension. Thebulk product is diluted in a final formulation appropriate for storageand use as described below. Containers are filled, and sealed with acontainer closure system and the drug product is labeled. The drugproduct is stored at an appropriate temperature to preserve stabilityand is tested for identity, strength, purity, potency, and quality andreleased for human use if it meets specified acceptance criteria.

2. Compositions

Pharmaceutically acceptable compositions are prepared in view ofapprovals for a regulatory agency or other agency prepared in accordancewith generally recognized pharmacopeia for use in animals and in humans.The compositions can be prepared as solutions, suspensions, powders, orsustained release formulations. Typically, the compounds are formulatedinto pharmaceutical compositions using techniques and procedures wellknown in the art (see e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Edition, 1985, 126). The formulation should suit the modeof administration.

Compositions can be formulated for administration by any route known tothose of skill in the art including intramuscular, intravenous,intradermal, intralesional, intraperitoneal injection, subcutaneous,intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local,otic, inhalational, buccal (e.g., sublingual), and transdermaladministration or any route. Other modes of administration also arecontemplated. Administration can be local, topical or systemic dependingupon the locus of treatment. Local administration to an area in need oftreatment can be achieved by, for example, but not limited to, localinfusion during surgery, topical application, e.g., in conjunction witha wound dressing after surgery, by injection, by means of a catheter, bymeans of a suppository, or by means of an implant. Compositions also canbe administered with other biologically active agents, eithersequentially, intermittently or in the same composition. Administrationalso can include controlled release systems including controlled releaseformulations and device controlled release, such as by means of a pump.

The most suitable route in any given case depends on a variety offactors, such as the nature of the disease, the progress of the disease,the severity of the disease and the particular composition which isused. Pharmaceutical compositions can be formulated in dosage formsappropriate for each route of administration. In particular, thecompositions can be formulated into any suitable pharmaceuticalpreparations for systemic, local intraperitoneal, oral or directadministration. For example, the compositions can be formulated foradministration subcutaneously, intramuscularly, intratumorally,intravenously or intradermally. Administration methods can be employedto decrease the exposure of the active agent to degradative processes,such as immunological intervention via antigenic and immunogenicresponses. Examples of such methods include local administration at thesite of treatment or continuous infusion.

The immunostimulatory bacteria can be formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administrations well as transdermalpatch preparation and dry powder inhalers. Typically, the compounds areformulated into pharmaceutical compositions using techniques andprocedures well known in the art (see e.g., Ansel Introduction toPharmaceutical Dosage Forms, Fourth Edition, 1985, 126). Generally, themode of formulation is a function of the route of administration. Thecompositions can be formulated in dried (lyophilized or other forms ofvitrification) or liquid form. Where the compositions are provided indried form they can be reconstituted just prior to use by addition of anappropriate buffer, for example, a sterile saline solution.

3. Formulations

a. Liquids, Injectables, Emulsions

The formulation generally is made to suit the route of administration.Parenteral administration, generally characterized by injection orinfusion, either subcutaneously, intramuscularly, intratumorally,intravenously or intradermally is contemplated herein. Preparations ofbacteria for parenteral administration include suspensions ready forinjection (direct administration) or frozen suspension that are thawedprior to use, dry soluble products, such as lyophilized powders, readyto be combined with a resuspension solution just prior to use, andemulsions. Dried thermostable formulations such as lyophilizedformulations can be used for storage of unit doses for later use.

The pharmaceutical preparation can be in a frozen liquid form, forexample a suspension. If provided in frozen liquid form, the drugproduct can be provided as a concentrated preparation to be thawed anddiluted to a therapeutically effective concentration before use.

The pharmaceutical preparations also can be provided in a dosage formthat does not require thawing or dilution for use. Such liquidpreparations can be prepared by conventional means with pharmaceuticallyacceptable additives, as appropriate, such as suspending agents (e.g.,sorbitol, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, or fractionated vegetable oils); andpreservatives suitable for use with microbial therapeutics. Thepharmaceutical preparations can be presented in dried form, such aslyophilized or spray-dried, for reconstitution with water or othersterile suitable vehicle before use.

Suitable excipients are, for example, water, saline, dextrose, orglycerol. The solutions can be either aqueous or non-aqueous. Ifadministered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and other buffered solutionsused for intravenous hydration. For intratumoral administrationsolutions containing thickening agents such as glucose, polyethyleneglycol, and polypropylene glycol, oil emulsions and mixtures thereof canbe appropriate to maintain localization of the injectant.

Pharmaceutical compositions can include carriers or other excipients.For example, pharmaceutical compositions provided herein can contain anyone or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s),coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s),preservative(s), detergent(s), or sorbent(s) and a combination thereofor vehicle with which a modified therapeutic bacteria is administered.For example, pharmaceutically acceptable carriers or excipients used inparenteral preparations include aqueous vehicles, non-aqueous vehicles,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Formulations,including liquid preparations, can be prepared by conventional meanswith pharmaceutically acceptable additives or excipients.

Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which the compositions areadministered. Examples of suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of thecompound or agent, generally in purified form or partially purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, and sesame oil. Water is a typical carrier.Saline solutions and aqueous dextrose and glycerol solutions also can beemployed as liquid carriers, particularly for injectable solutions.Compositions can contain along with an active ingredient: a diluent suchas lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; alubricant, such as magnesium stearate, calcium stearate and talc; and abinder such as starch, natural gums, such as gum acacia, gelatin,glucose, molasses, polyvinylpyrrolidine, celluloses and derivativesthereof, povidone, crospovidones and other such binders known to thoseof skill in the art. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, and ethanol. Forexample, suitable excipients are, for example, water, saline, dextrose,glycerol or ethanol. A composition, if desired, also can contain otherminor amounts of non-toxic auxiliary substances such as wetting oremulsifying agents, pH buffering agents, stabilizers, solubilityenhancers, and other such agents, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, non-aqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Non-aqueous parenteral vehicles includefixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Isotonic agents include sodium chloride and dextrose.Buffers include phosphate and citrate. Antioxidants include sodiumbisulfate. Local anesthetics include procaine hydrochloride. Suspendingand dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include, for example, polysorbates, such Polysorbate 80 (TWEEN80). Sequestering or chelating agents of metal ions, such as EDTA, canbe included. Pharmaceutical carriers also include polyethylene glycoland propylene glycol for water miscible vehicles and sodium hydroxide,hydrochloric acid, citric acid or lactic acid for pH adjustment.Non-anti-microbial preservatives can be included.

The pharmaceutical compositions also can contain other minor amounts ofnon-toxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, stabilizers, solubility enhancers, and other suchagents, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate and cyclodextrins. Implantation of a slow-releaseor sustained-release system, such that a constant level of dosage ismaintained (see, e.g., U.S. Pat. No. 3,710,795) also is contemplatedherein. The percentage of active compound contained in such parenteralcompositions is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject.

b. Dried Thermostable Formulations

The bacteria can be dried. Dried thermostable formulations, such aslyophilized or spray dried powders and vitrified glass can bereconstituted for administration as solutions, emulsions and othermixtures. The dried thermostable formulation can be prepared from any ofthe liquid formulations, such as the suspensions, described above. Thepharmaceutical preparations can be presented in lyophilized or vitrifiedform for reconstitution with water or other suitable vehicle before use.

The thermostable formulation is prepared for administration byreconstituting the dried compound with a sterile solution. The solutioncan contain an excipient which improves the stability or otherpharmacological attribute of the active substance or reconstitutedsolution, prepared from the powder. The thermostable formulation isprepared by dissolving an excipient, such as dextrose, sorbitol,fructose, corn syrup, xylitol, glycerin, glucose, sucrose or othersuitable agent, in a suitable buffer, such as citrate, sodium orpotassium phosphate or other such buffer known to those of skill in theart. Then, the drug substance is added to the resulting mixture, andstirred until it is mixed. The resulting mixture is apportioned intovials for drying. Each vial will contain a single dosage containing1×10⁵ to 1×10¹¹ CFUs per vial. After drying, the product vial is sealedwith a container closure system that prevents moisture or contaminantsfrom entering the sealed vial. The dried product can be stored underappropriate conditions, such as at −20° C., 4° C., or room temperature.Reconstitution of this dried formulation with water or a buffer solutionprovides a formulation for use in parenteral administration. The preciseamount depends upon the indication treated and selected compound. Suchamount can be empirically determined.

4. Compositions for Other Routes of Administration

Depending upon the condition treated, other routes of administration inaddition to parenteral, such as topical application, transdermalpatches, oral and rectal administration are also contemplated herein.The suspensions and powders described above can be administered orallyor can be reconstituted for oral administration. Pharmaceutical dosageforms for rectal administration are rectal suppositories, capsules andtablets and gel capsules for systemic effect. Rectal suppositoriesinclude solid bodies for insertion into the rectum which melt or softenat body temperature releasing one or more pharmacologically ortherapeutically active ingredients. Pharmaceutically acceptablesubstances in rectal suppositories are bases or vehicles and agents toraise the melting point. Examples of bases include cocoa butter(theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) andappropriate mixtures of mono-, di- and triglycerides of fatty acids.Combinations of the various bases can be used. Agents to raise themelting point of suppositories include spermaceti and wax. Rectalsuppositories can be prepared either by the compressed method or bymolding. The typical weight of a rectal suppository is about 2 to 3 gm.Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration. Formulations suitable forrectal administration can be provided as unit dose suppositories. Thesecan be prepared by admixing the drug substance with one or moreconventional solid carriers, for example, cocoa butter, and then shapingthe resulting mixture.

For oral administration, pharmaceutical compositions can take the formof, for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). The tabletscan be coated by methods well-known in the art.

Formulations suitable for buccal (sublingual) administration include,for example, lozenges containing the active compound in a flavored base,usually sucrose and acacia or tragacanth; and pastilles containing thecompound in an inert base such as gelatin and glycerin or sucrose andacacia.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixtures can be solutions, suspensions,emulsion or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compositions can be formulated as aerosols for topical application,such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126; 4,414,209and 4,364,923, which describe aerosols for delivery of a steroid usefulfor treatment of lung diseases). These formulations, for administrationto the respiratory tract, can be in the form of an aerosol or solutionfor a nebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will typically have diameters of less than50 microns, or less than 10 microns.

The compounds can be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients also can be administered.

Formulations suitable for transdermal administration are provided. Theycan be provided in any suitable format, such as discrete patches adaptedto remain in intimate contact with the epidermis of the recipient for aprolonged period of time. Such patches contain the active compound in anoptionally buffered aqueous solution of, for example, 0.1 to 0.2 Mconcentration with respect to the active compound. Formulations suitablefor transdermal administration also can be delivered by iontophoresis(see, e.g., Tyle, P, (1986) Pharmaceutical Research 3(6):318-326) andtypically take the form of an optionally buffered aqueous solution ofthe active compound.

Pharmaceutical compositions also can be administered by controlledrelease formulations and/or delivery devices (see e.g., U.S. Pat. Nos.3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,916,899; 4,008,719;4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,591,767; 5,639,476;5,674,533 and 5,733,566).

5. Dosages and Administration

The compositions can be formulated as pharmaceutical compositions forsingle dosage or multiple dosage administration. The immunostimulatorybacteria can be included in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the patient treated. For example, the concentration of thepharmaceutically active compound is adjusted so that an injectionprovides an effective amount to produce the desired pharmacologicaleffect. The therapeutically effective concentration can be determinedempirically by testing the immunostimulatory bacteria in known in vitroand in vivo systems such as by using the assays described herein orknown in the art. For example, standard clinical techniques can beemployed. In vitro assays and animal models can be employed to helpidentify optimal dosage ranges. The precise dose, which can bedeterminied empirically, can depend on the age, weight, body surfacearea, and condition of the patient or animal, the particularimmunostimulatory bacteria administered, the route of administration,the type of disease to be treated and the seriousness of the disease.

Hence, it is understood that the precise dosage and duration oftreatment is a function of the disease being treated and can bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. Concentrations and dosage valuesalso can vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or use of compositions and combinationscontaining them. The compositions can be administered hourly, daily,weekly, monthly, yearly or once. Generally, dosage regimens are chosento limit toxicity. It should be noted that the attending physician wouldknow how to and when to terminate, interrupt or adjust therapy to lowerdosage due to toxicity, or bone marrow, liver or kidney or other tissuedysfunctions. Conversely, the attending physician would also know how toand when to adjust treatment to higher levels if the clinical responseis not adequate (precluding toxic side effects).

The immunostimulatory bacteria are included in the composition in anamount sufficient to exert a therapeutically useful effect. For example,the amount is one that achieves a therapeutic effect in the treatment ofa hyperproliferative disease or condition, such as cancer.

Pharmaceutically and therapeutically active compounds and derivativesthereof are typically formulated and administered in unit dosage formsor multiple dosage forms. Each unit dose contains a predeterminedquantity of therapeutically active compound sufficient to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier, vehicle or diluent. Unit dosage forms, include,but are not limited to, tablets, capsules, pills, powders, granules,parenteral suspensions, and oral solutions or suspensions, and oil wateremulsions containing suitable quantities of the compounds orpharmaceutically acceptable derivatives thereof. Unit dose forms can becontained in vials, ampoules and syringes or individually packagedtablets or capsules. Unit dose forms can be administered in fractions ormultiples thereof. A multiple dose form is a plurality of identical unitdosage forms packaged in a single container to be administered insegregated unit dose form. Examples of multiple dose forms includevials, bottles of tablets or capsules or bottles of pints or gallons.Hence, multiple dose form is a multiple of unit doses that are notsegregated in packaging. Generally, dosage forms or compositionscontaining active ingredient in the range of 0.005% to 100% with thebalance made up from non-toxic carrier can be prepared. Pharmaceuticalcompositions can be formulated in dosage forms appropriate for eachroute of administration.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. The volume of liquid solution orreconstituted powder preparation, containing the pharmaceutically activecompound, is a function of the disease to be treated and the particulararticle of manufacture chosen for package. All preparations forparenteral administration must be sterile, as is known and practiced inthe art.

As indicated, compositions provided herein can be formulated for anyroute known to those of skill in the art including, but not limited to,subcutaneous, intramuscular, intravenous, intradermal, intralesional,intraperitoneal injection, epidural, vaginal, rectal, local, otic,transdermal administration or any route of administration. Formulationssuited for such routes are known to one of skill in the art.Compositions also can be administered with other biologically activeagents, either sequentially, intermittently or in the same composition.

Pharmaceutical compositions can be administered by controlled releaseformulations and/or delivery devices (see, e.g., U.S. Pat. Nos.3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899;4,008,719; 4,687,660; 4,769,027; 5,059,595; 5,073,543; 5,120,548;5,354,556; 5,591,767; 5,639,476; 5,674,533 and 5,733,566). Variousdelivery systems are known and can be used to administer selectedcompositions, are contemplated for use herein, and such particles can beeasily made.

6. Packaging and Articles of Manufacture

Also provided are articles of manufacture containing packagingmaterials, any pharmaceutical composition provided herein, and a labelthat indicates that the compositions are to be used for treatment ofdiseases or conditions as described herein. For example, the label canindicate that the treatment is for a tumor or cancer.

Combinations of immunostimulatory bacteria described herein and anothertherapeutic agent also can be packaged in an article of manufacture. Inone example, the article of manufacture contains a pharmaceuticalcomposition containing the immunostimulatory bacteria composition and nofurther agent or treatment. In other examples, the article ofmanufacture another further therapeutic agent, such as a differentanti-cancer agent. In this example, the agents can be provided togetheror separately, for packaging as articles of manufacture.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. Exemplary of articles of manufactureare containers including single chamber and dual chamber containers. Thecontainers include, but are not limited to, tubes, bottles and syringes.The containers can further include a needle for intravenousadministration.

The choice of package depends on the agents, and whether suchcompositions will be packaged together or separately. In general, thepackaging is non-reactive with the compositions contained therein. Inother examples, some of the components can be packaged as a mixture. Inother examples, all components are packaged separately. Thus, forexample, the components can be packaged as separate compositions that,upon mixing just prior to administration, can be directly administeredtogether. Alternatively, the components can be packaged as separatecompositions for administration separately.

Selected compositions including articles of manufacture thereof also canbe provided as kits. Kits can include a pharmaceutical compositiondescribed herein and an item for administration provided as an articleof manufacture. The compositions can be contained in the item foradministration or can be provided separately to be added later. The kitcan, optionally, include instructions for application including dosages,dosing regimens and instructions for modes of administration. Kits alsocan include a pharmaceutical composition described herein and an itemfor diagnosis.

I. Methods of Treatment and Uses

The methods provided herein include methods of administering or usingthe immunostimulatory bacteria, for treating subjects having a diseaseor condition whose symptoms can be ameliorated or lessened byadministration of such bacteria, such as cancer. In particular examples,the disease or condition is a tumor or a cancer. Additionally, methodsof combination therapies with one or more additional agents fortreatment, such as an anti-cancer agent, such as an oncolytic virus, animmunotherapeutic agent, and/or an anti-hyaluronan agent, such as ahyaluronidase, also are provided. The bacteria can be administered byany suitable route, including, but not limited to, parenteral, systemic,topical and local, such as intra-tumoral, intravenous, rectal, oral,intramuscular, mucosal and other routes. Because of the modifications ofthe bacteria described herein, problems associated with systemicadministration, are solved. Formulations suitable for each route ofadministration are provided. The skilled person can establish suitableregimens and doses and select routes.

1. Tumors

The immunostimulatory bacteria, combinations, uses and methods providedherein are applicable to treating all types of tumors, includingcancers, particularly solid tumors including lung cancer, bladdercancer, non-small cell lung cancer, gastric cancers, head and neckcancers, ovarian cancer, liver cancer, pancreatic cancer, kidney cancer,breast cancer, colorectal cancer, and prostate cancer. The methods alsocan be used for hematological cancers.

Tumors and cancers subject to treatment by the uses and methods providedherein include, but are not limited to, those that originate in theimmune system, skeletal system, muscles and heart, breast, pancreas,gastrointestinal tract, central and peripheral nervous system, renalsystem, reproductive system, respiratory system, skin, connective tissuesystems, including joints, fatty tissues, and circulatory system,including blood vessel walls. Examples of tumors that can be treatedwith the immunostimulatory bacteria provided herein include carcinomas,gliomas, sarcomas (including liposarcoma), adenocarcinomas,adenosarcomas, and adenomas. Such tumors can occur in virtually allparts of the body, including, for example, the breast, heart, lung,small intestine, colon, spleen, kidney, bladder, head and neck, ovary,prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus,uterus, testicles, cervix or liver.

Tumors of the skeletal system include, for example, sarcomas andblastomas such as osteosarcoma, chondrosarcoma, and chondroblastoma.Muscle and heart tumors include tumors of both skeletal and smoothmuscles, e.g., leiomyomas (benign tumors of smooth muscle),leiomyosarcomas, rhabdomyomas (benign tumors of skeletal muscle),rhabdomyosarcomas, and cardiac sarcoma. Tumors of the gastrointestinaltract include, e.g., tumors of the mouth, esophagus, stomach, smallintestine, colon and colorectal tumors, as well as tumors ofgastrointestinal secretory organs such as the salivary glands, liver,pancreas, and the biliary tract. Tumors of the central nervous systeminclude tumors of the brain, retina, and spinal cord, and can alsooriginate in associated connective tissue, bone, blood vessels ornervous tissue. Treatment of tumors of the peripheral nervous system arealso contemplated. Tumors of the peripheral nervous system includemalignant peripheral nerve sheath tumors. Tumors of the renal systeminclude those of the kidneys, e.g., renal cell carcinoma, as well astumors of the ureters and bladder. Tumors of the reproductive systeminclude tumors of the cervix, uterus, ovary, prostate, testes andrelated secretory glands. Tumors of the immune system include bothblood-based and solid tumors, including lymphomas, e.g., both Hodgkin'sand non-Hodgkin's. Tumors of the respiratory system include tumors ofthe nasal passages, bronchi and lungs. Tumors of the breast include,e.g., both lobular and ductal carcinoma.

Other examples of tumors that can be treated by the immunostimulatorybacteria and methods provided herein include Kaposi's sarcoma, CNSneoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas andcerebral metastases, melanoma, gastrointestinal and renal carcinomas andsarcomas, rhabdomyosarcoma, glioblastoma (such as glioblastomamultiforme) and leiomyosarcoma. Examples of other cancers that can betreated as provided herein include, but are not limited to, lymphoma,blastoma, neuroendocrine tumors, mesothelioma, schwannoma, meningioma,melanoma, and leukemia or lymphoid malignancies. Examples of suchcancers include hematologic malignancies, such as Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticlymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, Langerhans cellhistocytosis, myeloid neoplasias such as acute myelogenous leukemias,including AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia;tumors of the central nervous system such as glioma, glioblastoma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma; solid tumors of the head and neck (e.g., nasopharyngealcancer, salivary gland carcinoma, and esophageal cancer), lung (e.g.,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung and squamous carcinoma of the lung), digestive system (e.g.,gastric or stomach cancer including gastrointestinal cancer, cancer ofthe bile duct or biliary tract, colon cancer, rectal cancer, colorectalcancer, and anal carcinoma), reproductive system (e.g., testicular,penile, or prostate cancer, uterine, vaginal, vulval, cervical, ovarian,and endometrial cancer), skin (e.g., melanoma, basal cell carcinoma,squamous cell cancer, actinic keratosis, cutaneous melanoma), liver(e.g., liver cancer, hepatic carcinoma, hepatocellular cancer, andhepatoma), bone (e.g., osteoclastoma, and osteolytic bone cancers)additional tissues and organs (e.g., pancreatic cancer, bladder cancer,kidney or renal cancer, thyroid cancer, breast cancer, cancer of theperitoneum, and Kaposi's sarcoma), tumors of the vascular system (e.g.,angiosarcoma and hemangiopericytoma), Wilms' tumor, retinoblastoma,osteosarcoma and Ewing's sarcoma.

2. Administration

In practicing the uses and methods herein, immunostimulatory bacteriaprovided herein can be administered to a subject, including a subjecthaving a tumor or having neoplastic cells, or a subject to be immunized.One or more steps can be performed prior to, simultaneously with orafter administration of the immunostimulatory bacteria to the subjectincluding, but not limited to, diagnosing the subject with a conditionappropriate for administering immunostimulatory bacteria, determiningthe immunocompetence of the subject, immunizing the subject, treatingthe subject with a chemotherapeutic agent, treating the subject withradiation, or surgically treating the subject.

For embodiments that include administering immunostimulatory bacteria toa tumor-bearing subject for therapeutic purposes, the subject typicallyhas previously been diagnosed with a neoplastic condition. Diagnosticmethods also can include determining the type of neoplastic condition,determining the stage of the neoplastic conditions, determining the sizeof one or more tumors in the subject, determining the presence orabsence of metastatic or neoplastic cells in the lymph nodes of thesubject, or determining the presence of metastases of the subject.

Some embodiments of therapeutic methods for administeringimmunostimulatory bacteria to a subject can include a step ofdetermination of the size of the primary tumor or the stage of theneoplastic disease, and if the size of the primary tumor is equal to orabove a threshold volume, or if the stage of the neoplastic disease isat or above a threshold stage, an immunostimulatory bacterium isadministered to the subject. In a similar embodiment, if the size of theprimary tumor is below a threshold volume, or if the stage of theneoplastic disease is at or below a threshold stage, theimmunostimulatory bacterium is not yet administered to the subject; suchmethods can include monitoring the subject until the tumor size orneoplastic disease stage reaches a threshold amount, and thenadministering the immunostimulatory bacterium to the subject. Thresholdsizes can vary according to several factors, including rate of growth ofthe tumor, ability of the immunostimulatory bacterium to infect a tumor,and immunocompetence of the subject. Generally the threshold size willbe a size sufficient for an immunostimulatory bacterium to accumulateand replicate in or near the tumor without being completely removed bythe host's immune system, and will typically also be a size sufficientto sustain a bacterial infection for a time long enough for the host tomount an immune response against the tumor cells, typically about oneweek or more, about ten days or more, or about two weeks or more.Exemplary threshold stages are any stage beyond the lowest stage (e.g.,Stage I or equivalent), or any stage where the primary tumor is largerthan a threshold size, or any stage where metastatic cells are detected.

Any mode of administration of a microorganism to a subject can be used,provided the mode of administration permits the immunostimulatorybacteria to enter a tumor or metastasis. Modes of administration caninclude, but are not limited to, intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intratumoral, multipuncture,inhalation, intranasal, oral, intracavity (e.g., administering to thebladder via a catheter, administering to the gut by suppository orenema), aural, rectal, and ocular administration.

One skilled in the art can select any mode of administration compatiblewith the subject and the bacteria, and that also is likely to result inthe bacteria reaching tumors and/or metastases. The route ofadministration can be selected by one skilled in the art according toany of a variety of factors, including the nature of the disease, thekind of tumor, and the particular bacteria contained in thepharmaceutical composition. Administration to the target site can beperformed, for example, by ballistic delivery, as a colloidal dispersionsystem, or systemic administration can be performed by injection into anartery.

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. A single dose can be therapeutically effective for treating adisease or disorder in which immune stimulation effects treatment.Exemplary of such stimulation is an immune response, that includes, butis not limited to, one or both of a specific immune response andnon-specific immune response, both specific and non-specific responses,innate response, primary immune response, adaptive immunity, secondaryimmune response, memory immune response, immune cell activation, immunecell proliferation, immune cell differentiation, and cytokineexpression.

As is known in the medical arts, dosages for a subject can depend onmany factors, including the subject's species, size, body surface area,age, sex, immunocompetence, and general health, the particular bacteriato be administered, duration and route of administration, the kind andstage of the disease, for example, tumor size, and other compounds suchas drugs being administered concurrently. In addition to the abovefactors, such levels can be affected by the infectivity of the bacteriaand the nature of the bacteria, as can be determined by one skilled inthe art. In the present methods, appropriate minimum dosage levels ofbacteria can be levels sufficient for the bacteria to survive, grow andreplicate in a tumor or metastasis. Exemplary minimum levels foradministering a bacterium to a 65 kg human can include at least about5×10⁶ colony forming units (CFU), at least about 1×10⁷ CFU, at leastabout 5×10⁷ CFU, at least about 1×10⁸ CFU, or at least about 1×10⁹ CFU.In the present methods, appropriate maximum dosage levels of bacteriacan be levels that are not toxic to the host, levels that do not causesplenomegaly of 3× or more, levels that do not result in colonies orplaques in normal tissues or organs after about 1 day or after about 3days or after about 7 days. Exemplary maximum levels for administering abacterium to a 65 kg human can include no more than about 5×10¹¹ CFU, nomore than about 1×10¹¹ CFU, no more than about 5×10¹⁰ CFU, no more thanabout 1×10¹⁰ CFU, or no more than about 1×10⁹ CFU.

The methods and uses provided herein can include a single administrationof immunostimulatory bacteria to a subject or multiple administrationsof immunostimulatory bacteria to a subject or others of a variety ofregimens, including combination therapies with other anti-tumortherapeutics and/or treatments. These include, cellular therapies, suchas administration of modified immune cells; CAR-T therapy; CRISPRtherapy; immunotherapy, such as immune checkpoint inhibitors, such asantibodies and antibody fragments; chemotherapy and chemotherapeuticcompounds, such as nucleoside analogs; surgery; oncolytic virus therapy;and radiotherapy.

The immunostimulatory bacteria, or pharmaceutical compositionscontaining the immunostimulatory bacteria, can be used in methods oftreatment, wherein the treatment comprises combination therapy, in whicha second anti-cancer agent or treatment is administered. The secondanti-cancer agent or treatment is administered before, concomitantlywith, after, or intermittently with, the immunostimulatory bacterium orpharmaceutical composition, and can be an immunotherapy, oncolytic virustherapy, radiation, chemotherapy, or surgery, for example. Theimmunotherapy can be an antibody or antibody fragment, such as anantigen-binding fragment, including an anti-PD-1, or anti-PD-L1, oranti-CTLA4, or anti-IL6, or anti-VEGF, or anti-VEGFR, or anti-VEGFR2antibody, or fragments thereof.

In some embodiments, a single administration is sufficient to establishimmunostimulatory bacteria in a tumor, where the bacteria can colonizeand can cause or enhance an anti-tumor response in the subject. In otherembodiments, the immunostimulatory bacteria provided for use in themethods herein can be administered on different occasions, separated intime typically by at least one day. Separate administrations canincrease the likelihood of delivering a bacterium to a tumor ormetastasis, where a previous administration may have been ineffective indelivering the bacterium to a tumor or metastasis. In embodiments,separate administrations can increase the locations on a tumor ormetastasis where bacterial colonization/proliferation can occur or canotherwise increase the titer of bacteria accumulated in the tumor, whichcan increase eliciting or enhancing a host's anti-tumor immune response.

When separate administrations are performed, each administration can bea dosage amount that is the same or different relative to otheradministration dosage amounts. In one embodiment, all administrationdosage amounts are the same. In other embodiments, a first dosage amountcan be a larger dosage amount than one or more subsequent dosageamounts, for example, at least 10× larger, at least 100× larger, or atleast 1000× larger than subsequent dosage amounts. In one example of amethod of separate administrations in which the first dosage amount isgreater than one or more subsequent dosage amounts, all subsequentdosage amounts can be the same, smaller amount relative to the firstadministration.

Separate administrations can include any number of two or moreadministrations, including two, three, four, five or sixadministrations. One skilled in the art readily can determine the numberof administrations to perform, or the desirability of performing one ormore additional administrations, according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. Accordingly, the methods provided herein include methods ofproviding to the subject one or more administrations of aimmunostimulatory bacteria, where the number of administrations can bedetermined by monitoring the subject, and, based on the results of themonitoring, determining whether or not to provide one or more additionaladministrations. Deciding whether or not to provide one or moreadditional administrations can be based on a variety of monitoringresults, including, but not limited to, indication of tumor growth orinhibition of tumor growth, appearance of new metastases or inhibitionof metastasis, the subject's anti-bacterial antibody titer, thesubject's anti-tumor antibody titer, the overall health of the subjectand the weight of the subject.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response, the time period for a subject to clearbacteria from normal tissue, or the time period for bacterialcolonization/proliferation in the tumor or metastasis. In one example,the time period can be a function of the time period for a subject tomount an immune response; for example, the time period can be more thanthe time period for a subject to mount an immune response, such as morethan about one week, more than about ten days, more than about twoweeks, or more than about a month; in another example, the time periodcan be less than the time period for a subject to mount an immuneresponse, such as less than about one week, less than about ten days,less than about two weeks, or less than about a month. In anotherexample, the time period can be a function of the time period forbacterial colonization/proliferation in the tumor or metastasis; forexample, the time period can be more than the amount of time for adetectable signal to arise in a tumor or metastasis after administrationof a microorganism expressing a detectable marker, such as about 3 days,about 5 days, about a week, about ten days, about two weeks, or about amonth.

The methods used herein also can be performed by administeringcompositions, such as suspensions and other formulations, containing theimmunostimulatory bacteria provided herein. Such compositions containthe bacteria and a pharmaceutically acceptable excipient or vehicle, asprovided herein or known to those of skill in the art.

As discussed above, the uses and methods provided herein also caninclude administering one or more therapeutic compounds, such asanti-tumor compounds or other cancer therapeutics, to a subject inaddition to administering immunostimulatory bacteria to the subject. Thetherapeutic compounds can act independently, or in conjunction with theimmunostimulatory bacteria, for tumor therapeutic effects. Therapeuticcompounds that can act independently include any of a variety of knownchemotherapeutic compounds that can inhibit tumor growth, inhibitmetastasis growth and/or formation, decrease the size of a tumor ormetastasis, eliminate a tumor or metastasis, without reducing theability of the immunostimulatory bacteria to accumulate in a tumor,replicate in the tumor, and cause or enhance an anti-tumor immuneresponse in the subject. Examples of such chemotherapeutic agentsinclude, but are not limited to, alkylating agents such as thiotepa andcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin;antibiotics such as aclacinomycins, actinomycin, anthramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carubicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); anti-metabolites such as methotrexate and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; aziridines such as benzodepa, carboquone,meturedepa, and uredepa; ethylenimines and methyl-melamines includingaltretamine, triethyl enemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylol melamine; folic acidreplenisher such as folinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, chlorophos-phamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosoureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,such as paclitaxel and docetaxel and albuminated forms thereof (i.e.,nab-paclitaxel and nab-docetaxel), topoisomerase inhibitor RFS 2000;thymidylate synthase inhibitor (such as Tomudex); additionalchemotherapeutics including aceglatone; aldophosphamide glycoside;aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate;defosfamide; demecolcine; diaziquone; difluoromethylornithine (DMFO);eflornithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; Navelbine; Novantrone; teniposide; daunomycin; aminopterin;Xeloda; ibandronate; CPT-11; retinoic acid; esperamycins; capecitabine;and topoisomerase inhibitors such as irinotecan. Pharmaceuticallyacceptable salts, acids or derivatives of any of the above can also beused.

Therapeutic compounds that act in conjunction with the immunostimulatorybacteria include, for example, compounds that increase the immuneresponse eliciting properties of the bacteria, e.g., by increasingexpression of the RNAi, such as shRNA and miRNA, that inhibit, suppressor disrupt expression of the checkpoint genes, such as PD-L1, or TREX1or other checkpoint genes, or compounds that can further augmentbacterial colonization/proliferation. For example, a geneexpression-altering compound can induce or increase transcription of agene in a bacterium, such as an exogenous gene, e.g., encoding shRNAthat inhibit, suppress or disrupt expression of one or more checkpointgenes, thereby provoking an immune response. Any of a wide variety ofcompounds that can alter gene expression are known in the art, includingIPTG and RU486. Exemplary genes whose expression can be up-regulatedinclude proteins and RNA molecules, including toxins, enzymes that canconvert a prodrug to an anti-tumor drug, cytokines, transcriptionregulating proteins, shRNA, siRNA, and ribozymes. In other embodiments,therapeutic compounds that can act in conjunction with theimmunostimulatory bacteria to increase the colonization/proliferation orimmune response eliciting properties of the bacteria are compounds thatcan interact with a bacteria-expressed gene product, and suchinteraction can result in an increased killing of tumor cells or anincreased anti-tumor immune response in the subject. A therapeuticcompound that can interact with a bacteria-expressed gene product caninclude, for example a prodrug or other compound that has little or notoxicity or other biological activity in its subject-administered form,but after interaction with a bacteria-expressed gene product, thecompound can develop a property that results in tumor cell death,including but not limited to, cytotoxicity, ability to induce apoptosis,or ability to trigger an immune response. A variety of prodrug-likesubstances are known in the art, including ganciclovir, 5-fluorouracil,6-methylpurine deoxyriboside, cephalosporin-doxorubicin,4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,acetominophen, indole-3-acetic acid, CB1954,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycampotothecin,bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,epirubicin-glucoronide, 5′-deoxy5-fluorouridine, cytosine arabinoside,and linamarin.

3. Monitoring

The methods provided herein can further include one or more steps ofmonitoring the subject, monitoring the tumor, and/or monitoring theimmunostimulatory bacteria administered to the subject. Any of a varietyof monitoring steps can be included in the methods provided herein,including, but not limited to, monitoring tumor size, monitoring thepresence and/or size of metastases, monitoring the subject's lymphnodes, monitoring the subject's weight or other health indicatorsincluding blood or urine markers, monitoring anti-bacterial antibodytiter, monitoring bacterial expression of a detectable gene product, anddirectly monitoring bacterial titer in a tumor, tissue or organ of asubject.

The purpose of the monitoring can be simply for assessing the healthstate of the subject or the progress of therapeutic treatment of thesubject, or can be for determining whether or not further administrationof the same or a different immunostimulatory bacterium is warranted, orfor determining when or whether or not to administer a compound to thesubject where the compound can act to increase the efficacy of thetherapeutic method, or the compound can act to decrease thepathogenicity of the bacteria administered to the subject.

In some embodiments, the methods provided herein can include monitoringone or more bacterially expressed genes. Bacteria, such as thoseprovided herein or otherwise known in the art, can express one or moredetectable gene products, including but not limited to, detectableproteins.

As provided herein, measurement of a detectable gene product expressedin a bacterium can provide an accurate determination of the level ofbacteria present in the subject. As further provided herein, measurementof the location of the detectable gene product, for example, by imagingmethods including tomographic methods, can determine the localization ofthe bacteria in the subject. Accordingly, the methods provided hereinthat include monitoring a detectable bacterial gene product can be usedto determine the presence or absence of the bacteria in one or moreorgans or tissues of a subject, and/or the presence or absence of thebacteria in a tumor or metastases of a subject. Further, the methodsprovided herein that include monitoring a detectable bacterial geneproduct can be used to determine the titer of bacteria present in one ormore organs, tissues, tumors or metastases. Methods that includemonitoring the localization and/or titer of bacteria in a subject can beused for determining the pathogenicity of bacteria since bacterialinfection, and particularly the level of infection, of normal tissuesand organs can indicate the pathogenicity of the bacteria. The methodsthat include monitoring the localization and/or titer ofimmunostimulatory bacteria in a subject can be performed at multipletime points and, accordingly, can determine the rate of bacterialreplication in a subject, including the rate of bacterial replication inone or more organs or tissues of a subject; accordingly, methods thatinclude monitoring a bacterial gene product can be used for determiningthe replication competence of the bacteria. The methods provided hereinalso can be used to quantitate the amount of immunostimulatory bacteriapresent in a variety of organs or tissues, and tumors or metastases, andcan thereby indicate the degree of preferential accumulation of thebacteria in a subject; accordingly, the bacterial gene productmonitoring can be used in methods of determining the ability of thebacteria to accumulate in tumor or metastases in preference to normaltissues or organs. Since the immunostimulatory bacteria used in themethods provided herein can accumulate in an entire tumor or canaccumulate at multiple sites in a tumor, and can also accumulate inmetastases, the methods provided herein for monitoring a bacterial geneproduct can be used to determine the size of a tumor or the number ofmetastases present in a subject. Monitoring such presence of bacterialgene product in tumor or metastasis over a range of time can be used toassess changes in the tumor or metastases, including growth or shrinkingof a tumor, or development of new metastases or disappearance ofmetastases, and also can be used to determine the rate of growth orshrinking of a tumor, or development of new metastases or disappearanceof metastases, or the change in the rate of growth or shrinking of atumor, or development of new metastases or disappearance of metastases.Accordingly, monitoring a bacterial gene product can be used formonitoring a neoplastic disease in a subject, or for determining theefficacy of treatment of a neoplastic disease, by determining rate ofgrowth or shrinking of a tumor, or development of new metastases ordisappearance of metastases, or the change in the rate of growth orshrinking of a tumor, or development of new metastases or disappearanceof metastases.

Any of a variety of detectable proteins can be detected by monitoring,exemplary of which are any of a variety of fluorescence proteins (e.g.,green fluorescence proteins), any of a variety of luciferases,transferring or other iron binding proteins; or receptors, bindingproteins, and antibodies, where a compound that specifically binds thereceptor, binding protein or antibody can be a detectable agent or canbe labeled with a detectable substance (e.g., a radionuclide or imagingagent).

Tumor and/or metastasis size can be monitored by any of a variety ofmethods known in the art, including external assessment methods ortomographic or magnetic imaging methods. In addition to the methodsknown in the art, methods provided herein, for example, monitoringbacterial gene expression, can be used for monitoring tumor and/ormetastasis size.

Monitoring size over several time points can provide informationregarding the increase or decrease in size of a tumor or metastasis, andcan also provide information regarding the presence of additional tumorsand/or metastases in the subject. Monitoring tumor size over severaltime points can provide information regarding the development of aneoplastic disease in a subject, including the efficacy of treatment ofa neoplastic disease in a subject.

The methods provided herein also can include monitoring the antibodytiter in a subject, including antibodies produced in response toadministration of immunostimulatory bacteria to a subject. The bacteriaadministered in the methods provided herein can elicit an immuneresponse to endogenous bacterial antigens. The bacteria administered inthe methods provided herein also can elicit an immune response toexogenous genes expressed by the bacteria. The bacteria administered inthe methods provided herein also can elicit an immune response to tumorantigens. Monitoring antibody titer against bacterial antigens,bacterially expressed exogenous gene products, or tumor antigens can beused to monitor the toxicity of the bacteria, monitoring the efficacy oftreatment methods, or monitoring the level of gene product or antibodiesfor production and/or harvesting.

Monitoring antibody titer can be used to monitor the toxicity of thebacteria. Antibody titer against a bacteria can vary over the timeperiod after administration of the bacteria to the subject, where atsome particular time points, a low anti-(bacterial antigen) antibodytiter can indicate a higher toxicity, while at other time points a highanti-(bacterial antigen) antibody titer can indicate a higher toxicity.The bacteria used in the methods provided herein can be immunogenic, andcan, therefore, elicit an immune response soon after administering thebacteria to the subject. Generally, immunostimulatory bacteria againstwhich the immune system of a subject can mount a strong immune responsecan be bacteria that have low toxicity when the subject's immune systemcan remove the bacteria from all normal organs or tissues. Thus, in someembodiments, a high antibody titer against bacterial antigens soon afteradministering the bacteria to a subject can indicate low toxicity of thebacteria.

In other embodiments, monitoring antibody titer can be used to monitorthe efficacy of treatment methods. In the methods provided herein,antibody titer, such as anti-(tumor antigen) antibody titer, canindicate the efficacy of a therapeutic method such as a therapeuticmethod to treat neoplastic disease. Therapeutic methods provided hereincan include causing or enhancing an immune response against a tumorand/or metastasis. Thus, by monitoring the anti-(tumor antigen) antibodytiter, it is possible to monitor the efficacy of a therapeutic method incausing or enhancing an immune response against a tumor and/ormetastasis.

In other embodiments, monitoring antibody titer can be used formonitoring the level of gene product or antibodies for production and/orharvesting. As provided herein, methods can be used for producingproteins, RNA molecules or other compounds, particularly RNA moleculessuch as shRNA, by expressing an exogenous gene in a microorganism thathas accumulated in a tumor. Monitoring antibody titer against theprotein, RNA molecule or other compound can indicate the level ofproduction of the protein, RNA molecule or other compound by thetumor-accumulated microorganism, and also can directly indicate thelevel of antibodies specific for such a protein, RNA molecule or othercompound.

The methods provided herein also can include methods of monitoring thehealth of a subject. Some of the methods provided herein are therapeuticmethods, including neoplastic disease therapeutic methods. Monitoringthe health of a subject can be used to determine the efficacy of thetherapeutic method, as is known in the art. The methods provided hereinalso can include a step of administering to a subject animmunostimulatory bacterium, as provided herein. Monitoring the healthof a subject can be used to determine the pathogenicity of animmunostimulatory bacterium administered to a subject. Any of a varietyof health diagnostic methods for monitoring disease such as neoplasticdisease, infectious disease, or immune-related disease can be monitored,as is known in the art. For example, the weight, blood pressure, pulse,breathing, color, temperature or other observable state of a subject canindicate the health of a subject. In addition, the presence or absenceor level of one or more components in a sample from a subject canindicate the health of a subject. Typical samples can include blood andurine samples, where the presence or absence or level of one or morecomponents can be determined by performing, for example, a blood panelor a urine panel diagnostic test. Exemplary components indicative of asubject's health include, but are not limited to, white blood cellcount, hematocrit, and c-reactive protein concentration.

The methods provided herein can include monitoring a therapy, wheretherapeutic decisions can be based on the results of the monitoring.Therapeutic methods provided herein can include administering to asubject immunostimulatory bacteria, where the bacteria canpreferentially accumulate in a tumor and/or metastasis, and where thebacteria can cause or enhance an anti-tumor immune response. Suchtherapeutic methods can include a variety of steps including multipleadministrations of a particular immunostimulatory bacterium,administration of a second immunostimulatory bacterium, oradministration of a therapeutic compound. Determination of the amount,timing or type of immunostimulatory bacteria or compound to administerto the subject can be based on one or more results from monitoring thesubject. For example, the antibody titer in a subject can be used todetermine whether or not it is desirable to administer animmunostimulatory bacterium and, optionally, a compound, the quantity ofbacteria and/or compound to administer, and the type of bacteria and/orcompound to administer, where, for example, a low antibody titer canindicate the desirability of administering an additionalimmunostimulatory bacterium, a different immunostimulatory bacterium,and/or a therapeutic compound such as a compound that induces bacterialgene expression or a therapeutic compound that is effective independentof the immunostimulatory bacteria.

In another example, the overall health state of a subject can be used todetermine whether or not it is desirable to administer animmunostimulatory bacterium and, optionally, a compound, the quantity ofbacterium or compound to administer, and the type of bacterium and/orcompound to administer where, for example, determining that the subjectis healthy can indicate the desirability of administering additionalbacteria, different bacteria, or a therapeutic compound such as acompound that induces bacterial gene (e.g., shRNA that inhibits one ormore checkpoint gene(s)) expression. In another example, monitoring adetectable bacterially expressed gene product can be used to determinewhether it is desirable to administer an immunostimulatory bacteriumand, optionally, a compound, the quantity of bacterium and/or compoundto administer, and the type of bacterium and/or compound to administerwhere, for example, determining that the subject is healthy can indicatethe desirability of administering additional bacteria, differentbacteria, or a therapeutic compound such as a compound that inducesbacterial gene (e.g., shRNA that inhibits one or more checkpointgene(s)) expression. Such monitoring methods can be used to determinewhether or not the therapeutic method is effective, whether or not thetherapeutic method is pathogenic to the subject, whether or not thebacteria have accumulated in a tumor or metastasis, and whether or notthe bacteria have accumulated in normal tissues or organs. Based on suchdeterminations, the desirability and form of further therapeutic methodscan be derived.

In another example, monitoring can determine whether or notimmunostimulatory bacteria have accumulated in a tumor or metastasis ofa subject. Upon such a determination, a decision can be made to furtheradminister additional bacteria, a different immunostimulatory bacteriumand, optionally, a compound to the subject.

J. Example S

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Summary of Some Exemplary Engineered Immunostimulatory Bacterial Strainsand Nomenclature:

Strain Strain # Plasmid Background RNAi Targets Alternate name AST-100None YS1646 none VNP 20009 AST-101 None YS1646-ASD none ASD (asd geneknockout) AST-102 pEQU6 YS1646 none YS1646 (pEQU6-plasmid) AST-103 pEQU6YS1646 Scrambled YS1646 (pEQU6-shSCR) (shRNA) AST-104 pEQU6 YS1646muTREX1 YS1646 (pEQU6-shTREX1) (shRNA) ARI-108 AST-105 pEQU6 YS1646muPD-L1 YS1646 (pEQU6-shPDL1) (shRNA) ARI-115 AST-106 pEQU6 YS1646muTREX1 YS1646 (pEQU6-miTREX1) (microRNA) ARI-203 AST-107 pATI-U6YS1646-ASD Scrambled ASD (pATI-shSCR) (shRNA) AST-108 pATI-U6 YS1646-ASDmuTREX1 ASD (pATI-shTREX1) (shRNA) ARI-108 AST-109 pATIKAN-U6 YS1646-ASDScrambled ASD (pATIKan-shSCR) (shRNA) AST-110 pATIKAN-U6 YS1646-ASDmuTREX1 ASD (pATIKan-shTREX1) (shRNA) ARI-108 AST-111 None YS1646-ASD-None ASD/FLG (asd and flagellin knockout) fljb-fliC AST-112 pATI-U6YS1646-ASD- muTREX1 ASD/FLG (pATI-shTREX1) fljb-fliC (shRNA) ARI-108AST-113 pATI-U6 YS1646-ASD- muTREX1 ASD/FLG (pATI-U6 Kan fljb-fliC(shRNA) shTREX1) ARI-108 AST-114 None YS1646-ASD- None ASD/LLO (asdknockout/ LLO cytoLLO knock-in) AST-115 pATI-U6 YS1646-ASD- muTREX1ASD/LLO (pATIKan-shTREX1) LLO (shRNA) ARI-108 AST-116 pATIKanpBRori-U6YS1646-ASD Scrambled ASD (pATIKanLow-shSCR) AST-117 pATIKanpBRori-U6YS1646-ASD muTREX1 ASD (pATIKanLow-shTREX1) (shRNA) ARI-108 AST-118pATIKanpBRori-U6 YS1646-ASD- muTREX1 ASD/FLG (pATIKanLow-shTREX1)fljb-fliC (shRNA) ARI-108 AST-119 pATIKanpBRori-U6 YS1646-ASD- muTREX1ASD/LLO (pATIKanLow-shTREX1) pMTL-LLO (shRNA) ARI-108 AST-120 pEQU6YS1646-ASD- muTREX1 ASD/LLO (pEQU6-miTREX1) pMTL-LLO (microRNA) SuicidalARI-203 AST-121 pEQU6 YS1646 muVISTA YS1646 (pEQU6-shVISTA) ARI-157AST-122 pEQU6 YS1646 muTGF-beta YS1646 (pEQU6-TGF-beta) ARI-149 AST-123pEQU6 YS1645 muBeta-Catenin YS1646 (pEQU6-Beta-Catenin) ARI-166

It is understood that these strains are listed for reference, the samedeletions and insertions can be effected in a wild-type Salmonellatyphimurium strain, such as the strain deposited under ATCC accession#14028, or a strain having all of the identifying characteristicsthereof. The wild-type strain can additionally be made auxotrophic foradenosine by appropriate selection or deletions. The construction of anduse of these strains is described in Published International PCTApplication No. WO 2019/014398, and in U.S. Application Publication No.2019/0017050.

Example 1 Salmonella asd Gene Knockout Strain Engineering

Strain AST-101 was prepared. It is an attenuated Salmonella typhimuriumstrain derived from strain YS1646 (which can be purchased from ATCC,Catalog #202165) that has been engineered to be asd⁻ (an asd geneknockout). In this example, the Salmonella typhimurium strain YS1646asd⁻ gene deletion was engineered using modifications of the method ofDatsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)) asoutlined in FIG. 1, and described below. The methods and resultingproducts in this example and all examples below can be used with otherstarting bacteria, such as wild-type Salmonella typhimurium, such as thestrain deposited under ATCC accession #14028.

Introduction of the Lambda Red Helper Plasmid into YS1646 The YS1646strain was prepared to be electrocompetent as described previously(Sambrook J., (1998) Molecular Cloning, A Laboratory Manual, 2nd Ed.Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory) by growing aculture in LB and concentrating 100-fold and washing three times withice-cold 10% glycerol. The electrocompetent strain was electroporatedwith the Lambda red helper plasmid pKD46 (SEQ ID NO:218) using a 0.2 cmgap cuvette at the following settings: 2.5 kV, 186 ohms, 50 pF.Transformants carrying pKD46 were grown in 5 mL SOC medium withampicillin and 1 mM L-arabinose at 30° C. and selected on LB agar platescontaining ampicillin. A YS1646 clone containing the lambda red helperplasmid pKD46 then was made electrocompetent, as described above forYS1646.

Construction of Asd Gene Knockout Cassette

The asd gene from the genome of YS1646 (Broadway et al. (2014) J.Biotechnology 192:177-178) was used for designing the asd gene knockoutcassette. A plasmid containing 204 and 203 bp of homology to the lefthand and right hand regions, respectively, of the asd gene, wastransformed into DH5-alpha competent cells. A kanamycin gene cassetteflanked by lox P sites was cloned into this plasmid. The asd geneknockout cassette then was PCR amplified using primers asd−1 and asd−2(Table 1) and gel purified.

Execution of Asd Gene Deletion

The YS1646 strain carrying plasmid pKD46 was electroporated with thegel-purified linear asd gene knock-out cassette. Electroporated cellswere recovered in SOC medium and plated onto LB Agar plates supplementedwith Kanamycin (20 μg/mL) and diaminopimelic acid (DAP, 50 μg/ml).During this step, lambda red recombinase induces homologousrecombination of the chromosomal asd gene with the kan cassette (due tothe presence of homologous flanking sequences upstream and downstream ofthe chromosomal asd gene), and knockout of the chromosomal copy of theasd gene occurs. The presence of the disrupted asd gene in the selectedkanamycin resistant clones was confirmed by PCR amplification withprimers from the YS1646 genome flanking the sites of disruption (primerasd-3) and from the multi-cloning site (primer scFv-3) (Table 1).Colonies were also replica plated onto LB plates with and withoutsupplemental DAP to demonstrate DAP auxotrophy. All clones with the asdgene deletion were unable to grow in the absence of supplemental DAP,demonstrating DAP auxotrophy.

TABLE 1 Primer information Primer SEQ name Primer sequence ID NO. asd-1ccttcctaacgcaaattccctg 219 asd-2 ccaatgctctgcttaactcctg 220 asd-3gcctcgccatgtttcagtacg 221 asd-4 ggtctggtgcattccgagtac 222 scFv-3cataatctgggtccttggtctgc 223Kanamycin Gene Cassette Removal The kan selectable marker was removed byusing the Cre/loxP site-specific recombination system. The YS1646 asd⁻gene Kan^(R) mutant was transformed with pJW 168 (a temperaturesensitive plasmid expressing the cre recombinase, SEQ ID NO:224).Amp^(R) colonies were selected at 30° C.; pJW168 was subsequentlyeliminated by growth at 42° C. A selected clone (AST-101) then wastested for loss of kan by replica plating on LB agar plates with andwithout kanamycin, and confirmed by PCR verification using primers fromYS1646 genome flanking the sites of disruption (primer asd-3 and asd-4,for primer sequence, see Table 1).

Characterization of the Asd Deletion Mutant Strain AST-101

The asd mutant AST-101 was unable to grow on LB agar plates at 37° C.,but was able to grow on LB plates containing 50 μg/mL diaminopimelicacid (DAP). The asd mutant growth rate was evaluated in LB liquid mediaand it was unable to grow in liquid LB but was able to grow in LBsupplemented with 50 μg/mL DAP, as determined by measuring absorbance at600 nM.

Sequence Confirmation of the AST-101 Asd Locus Sequence after Asd GeneDeletion

The AST-101 asd gene deletion strain was verified by DNA sequencingusing primers asd-3 and asd-4. Sequencing of the region flanking the asdlocus was performed and the sequence confirmed that the asd gene wasdeleted from the YS1646 chromosome.

Example 2 Generation of Modified Salmonella typhimurium Strains fromWild-Type Salmonella typhimurium

The purI, msbB and asd genes were individually deleted from the genomeof wild-type Salmonella typhimurium strain ATCC 14028 using thelambda-derived Red recombination system as described in Datsenko andWanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), to generate abase-strain designated 14028:ΔpurI/ΔmsbB/Δasd. The flagellin genes fljBand fliC were subsequently deleted to generate the strain14028:ΔpurI/ΔmsbB/Δasd/ΔfljB/ΔfjiC, and the pagP gene was then deletedto generate the strain 14028: ΔpurI/ΔmsbB/Δasd/ΔfljB/ΔfliC/ΔpagP.Strains 14028:ΔpurI/ΔmsbB/Δasd/ΔfljB/ΔfliC and14028:ΔpurI/ΔmsbB/Δasd/ΔfljB/AfiC/ΔpagP were electroporated with aplasmid containing a functional asd gene, to complement the chromosomaldeletion of asd and ensure plasmid maintenance in vivo, and a eukaryoticexpression cassette encoding the red fluorescent protein mCherry undercontrol of the EF1-α promoter.

Example 3 Modified Salmonella typhimurium Targets Demonstrate RobustTumor Growth Inhibition in Multiple Syngeneic Murine Tumor Models

PD-L1

The immune system has evolved several checks and balances to limitautoimmunity. Programmed cell death protein 1 (PD-1) and programmeddeath-ligand 1 (PD-L1) are two examples of numerous inhibitory “immunecheckpoints,” which function by downregulating immune responses. Thebinding of PD-L1 to PD-1 interferes with CD8⁺ T cell signaling pathways,impairing the proliferation and effector function of CD8⁺ T cells, andinducing T cell tolerance (Topalian et al. (2012) N. Engl. J. Med.366:3443-3447).

Tumor colonization of a modified Salmonella typhimurium straindelivering shRNA to knockdown the PD-L1 gene disrupts its binding toPD-1, and its inhibition of CD8⁺ T cell function. PD-L1/PD-1 checkpointinhibition synergizes well with the immunostimulatory S. typhimuriumcontaining CpG plasmid DNA, all in one therapeutic modality. In place ofan RNAi, the immunostimulatory bacterium can be modified to encode anantigen-binding fragment or single chain antibody that inhibits PD-L1 orPD-1, to inhibit the PD-1 pathway.

To demonstrate the in vivo efficacy of the YS1646 strain containing aplasmid encoding shRNA against PD-L1 (AST-105), or other inhibitor ofPD-L1 or the PD-1 pathway, this strain, in comparison to the AST-102strain (containing a control plasmid that also contains CpG motifs) wasevaluated in a murine colon carcinoma model. For this experiment, 6-8week-old female BALB/c mice (10 mice per group) were inoculated SC inthe right flank with CT26 murine colon carcinoma (2×10⁵ cells in 100 μLPBS). Mice bearing established flank tumors were intravenously (IV)injected twice, four days apart, with 5×10⁶ CFUs of AST-105 or AST-102,or were IV administered an anti-PD-L1 antibody (4 mg/kg, BioXCell clone10F.9G2). Six hours following the first IV dose, mice were bled, andplasma was collected and assessed for pro-inflammatory cytokines usingthe Mouse Inflammation Cytometric Bead Array kit and analyzed by FACS(BD Biosciences).

Treatment with strain AST-105 demonstrated statistically significanttumor control compared to treatment with the plasmid-containing controlstrain AST-102 (69% TGI, p=0.05, day 25). Tumor growth inhibition wasalso greater for treatment with AST-105 (expressing shPD-L1) than fromsystemic administration of an anti-PD-L1 antibody (68% TGI vs.anti-PD-L1).

Comparing the production of innate pro-inflammatory cytokines at 6 hourspost IV injection, the cytokines elicited by strain AST-105 weresignificantly higher compared to the anti-PD-L1 antibody (p<0.05), andmuch higher than those from AST-102. These data demonstrate thatinhibiting PD-L1 within the tumor microenvironment, compared to systemicadministration of anti-PD-L1 antibody, uniquely activates potentpro-inflammatory cytokines that induce anti-tumor immunity and promotetumor growth inhibition in a murine model of colon carcinoma.

Example 4 Intratumoral Administration of Modified S. Typhimurium Shtrex1Provides Distal Tumor Colonization and Complete Anti-Tumor Responses ina Dual Flank Murine Colon Carcinoma Model

A hallmark of inducing adaptive immunity to a tumor is the ability toinduce regression of a distal, untreated tumor. To assess the ability ofthe YS1646 strain containing the pEQU6 shRNA plasmids to induce primaryand distal tumor growth inhibition in a dual flank murine coloncarcinoma model, 6-8 week-old female BALB/c mice (10 mice per group)were inoculated SC in the right and left flanks with CT26 murine coloncarcinoma (2×10⁵ cells in 100 μL PBS). Mice bearing established flanktumors were intratumorally (IT) injected twice, four days apart, intothe right flank tumor with 5×10⁶ CFUs of AST-104, (pEQU6 shTREX1 inYS1646), AST-105 (pEQU6 shPD-L1 in YS1646) or AST-102 (plasmid controlin YS1646), and compared to PBS control.

IT injection of AST-104 and AST-105 induced significant tumor growthinhibition in the injected tumor, compared to the PBS control(AST-105-60.5% TGI, p=0.03; AST-104-61.4% TGI, p=0.03 day 25). UnlikeAST-105, only AST-104 induced significant growth inhibition of thedistal, untreated tumor compared to PBS (60% TGI, p<0.0001, day 25), andsignificant distal tumor growth inhibition compared to AST-102containing the plasmid control (p=0.004, day 25). The AST-104 strainalso demonstrated significant tumor regression and increased survivalcompared to PBS control (p=0.0076, Log-rank (Mantel-Cox) test) with 2/10complete remissions.

To determine whether the bacteria colonize injected, as well as distaltumors, tumor-bearing mice treated with AST-104 were sacrificed andtumors were collected. Injected and distal tumors were transferred to Mtubes and were homogenized in PBS using a gentleMACS™ Dissociator(Miltenyi Biotec). Tumor homogenates were serially diluted and plated onLB agar plates and incubated at 37° C. for colony forming unit (CFU)determination. As shown in FIG. 2, the distal tumor was colonized to thesame extent as the injected tumor, indicating that the engineeredSalmonella strains dosed with an intratumoral route of administrationare able to transit and colonize distal lesions. These data demonstratethe potency of administering an immunostimulatory bacteriaintratumorally with the ability to systemically colonize distal tumorlesions preferentially over other organs, and the potency of activatingthe STING Type I Interferon pathway, leading to systemic tumorregression and complete remission.

Example 5 Modified S. typhimurium Strains with Plasmids Containing CpGElements Demonstrate Enhanced Anti-Tumor Activity Compared To YS1646Parental Strain

Toll-like receptors (TLRs) are key receptors for sensingpathogen-associated molecular patterns (PAMPs) and activating innateimmunity against pathogens (Akira et al. (2001) Nat. Immunol.2(8):675-680). Of these, TLR9 is responsible for recognizinghypomethylated CpG motifs in pathogenic DNA which do not occur naturallyin mammalian DNA (McKelvey et al. (2011) J. Autoimmunity 36:76).Recognition of CpG motifs upon phagocytosis of pathogens into endosomesin immune cell subsets induces IFR7-dependent type I interferonsignaling and activates innate and adaptive immunity. It is shownherein, that the S. typhimurium strain YS1646 carrying modifiedSalmonella typhimurium plasmids containing CpG motifs (YS1646 pEQU6Scramble) similarly activate TLR9 and induce type I IFN-mediated innateand adaptive immunity, as compared to the YS1646 strain without aplasmid.

The CpG motifs in the engineered plasmids used here are shown in Table2. The pEQU6 shSCR (non-cognate shRNA) plasmid in strain AST-103possesses 362 CpG motifs, indicating that Salmonella-based plasmiddelivery can be immuno-stimulatory and have an anti-tumor effect, whencompared to the same Salmonella lacking transformation with thisplasmid. To assess the ability of CpG-containing plasmids within YS1646to induce tumor growth inhibition in a murine colon carcinoma model, 6-8week-old female BALB/c mice (9 mice per group) were inoculatedsubcutaneously (SC) in the right flank with CT26 (2×10⁵ cells in 100 μLPBS). Mice bearing established flank tumors were IV injected weekly withthree doses of 5×10⁶ CFUs of YS1646 (AST-100) or YS1646 containing anshRNA scrambled plasmid with CpG motifs (AST-103), and compared to PBScontrol.

TABLE 2 CpG motifs in the engineered plasmids Number SEQ of CpG IDSequence name Motifs NO. pBR322 Origin 80 243 pEQU6 (shSCR) 362 244 AsdGene ORF 234 242 pATI-2.0 538 245

As shown in FIG. 3, the YS1646 (AST-100) strain demonstrated modesttumor control (32% TGI, p=ns, day 28) as compared to PBS. The AST-103strain, that varies from YS1646 only by the addition of theCpG-containing plasmid encoding a non-cognate scrambled shRNA,demonstrated highly significant tumor growth inhibition compared toYS1646 alone, untransformed and therefore lacking a plasmid (p=0.004,day 32).

The asd gene possesses 234 CpG motifs (Table 2), indicating that aplasmid containing it can have immunostimulatory properties. As shown inFIG. 16, AST-109 (YS1646-ASD with scrambled shRNA) had 51% tumor growthinhibition vs PBS alone, indicative of a strong immuno-stimulatoryeffect.

These data demonstrate the potent immunostimulatory properties ofplasmid DNA containing TLR9-activating CpG motifs within atumor-targeting attenuated strain of S. typhimurium.

Example 6 Vector Synthesis

Complementation of asd Deletion by asd Expression from Plasmids

A plasmid (pATIU6) was chemically synthesized and assembled (SEQ IDNO:225). The plasmid contained the following features: a high copy(pUC19) origin or replication, a U6 promoter for driving expression of ashort hairpin, an ampicillin resistance gene flanked by HindIIIrestriction sites for subsequent removal, and the asd gene containing 85base pairs of sequence upstream of the start codon (SEQ ID NO:246). Intothis vector, shRNAs targeting murine TREX1 or a scrambled, non-cognateshRNA sequence were introduced by restriction digestion with SpeI andXhoI and ligation and cloning into E. coli DH5-alpha. The resultingplasmids, designated pATI-shTREX1 and pATI-shSCR, respectively, wereamplified in E. coli and purified for transformation into the asdknockout strain AST-101 by electroporation and clonal selection on LBamp plates to produce strains AST-108, and AST-107, respectively. asd⁻mutants complemented with pATIU6-derived plasmids were able to grow onLB agar and liquid media in the absence of DAP.

In a subsequent iteration, the ampicillin resistance gene (AmpR) frompATI-shTREX1 was replaced with a kanamycin resistance gene. This wasaccomplished by digestion of pATI-shTREX1 plasmid with HindIII followedby gel purification to remove the AmpR gene. PCR amplification of thekanamycin resistance (KanR) gene using primers APR-001 and APR-002 (SEQID NO:226 and SEQ ID NO:227), digestion with HindIII and ligation intothe gel purified, digested pATIU6 plasmid.

In subsequent iterations, a single point mutation was introduced intothe pATIKan plasmid at the pUC 19 origin of replication using the Q5®Site-Directed Mutagenesis Kit (New England Biolabs) and the primersAPR-003 (SEQ ID NO:228) and APR-004 (SEQ ID NO:229) to change thenucleotide T at position 148 to a C. This mutation makes the origin ofreplication homologous to the pBR322 origin of replication in order toreduce the plasmid copy number.

Primer SEQ ID Description Sequence ID NO APR-001 Kan primerFAAAAAAGCTTGCAGCTCT 226 GGCCCGTG APR-002 Kan PrimerR AAAAAAGCTTTTAGAAAA227 ACTCATCGAGCATCAAAT GA APR-003 pATI ori ACACTAGAAGgACAGTAT 228 T148CFTTGGTATCTG APR-004 pATI ori AGCCGTAGTTAGGCCACC 229 T148CRpATI2.0

A plasmid was designed and synthesized that contains the followingfeatures: a pBR322 origin of replication, an SV40 DNA nuclear targetingsequence (DTS), an rrnB terminator, a U6 promoter for driving expressionof shRNAs followed by flanking restriction sites for cloning thepromoter and shRNAs or microRNAs, the asd gene, an rrnG terminator, anda kanamycin resistance gene flanked by HindIII sites for curing and amulticloning site (SEQ ID NO:247). In addition, a plasmid was designedand synthesized for expression of two separate shRNA or microRNAs. Thisplasmid contains the following features: a pBR322 origin of replication,an SV40 DNA nuclear targeting sequence (DTS), an rrnB terminator, a U6promoter for driving expression of shRNAs followed by flankingrestriction sites for cloning the promoter and shRNAs or microRNAs, anH1 promoter for driving the expression of a 2^(nd) shRNA or microRNA, a450 bp randomly generated stuffer sequence placed between the H1 and U6promoters, the asd gene, an rrnG terminator, and a kanamycin resistancegene flanked by HindIII sites for curing and a multicloning site (SEQ IDNO:245).

Example 7 S. typhimurium Flagellin Knockout Strain Engineering byDeletion of the fliC and fljB Genes

In the example herein, S. typhimurium strains were engineered to lackboth flagellin subunits fliC and fljB to reduce pro-inflammatorysignaling. Deletions of fliC and fljB were sequentially engineered intothe chromosome of the asd gene-deleted strain of YS1646 (AST-101).

Deletion of fliC

In this example, fliC was deleted from the chromosome of the AST-101strain using modifications of the method of Datsenko and Wanner (Proc.Natl. Acad. Sci. USA 97:6640-6645 (2000)) as described in detail inExample 1 and schematically depicted in FIG. 4. Synthetic fliC genehomology arm sequences were ordered that contained 224 and 245 bases ofhomologous sequence flanking the fliC gene, cloned into a plasmid calledpSL0147 (SEQ ID NO:230). A kanamycin gene cassette flanked by cre/lox psites then was cloned into pSL0147, the fliC gene knockout cassette wasthen PCR amplified with primer flic-1 (SEQ ID NO:232) and flic-2 (SEQ IDNO:233) and gel purified and introduced into the AST-101 strain carryingthe temperature sensitive lambda red recombination plasmid pKD46 byelectroporation. Electroporated cells were recovered in SOC+DAP mediumand plated onto LB Agar plates supplemented with Kanamycin (20 μg/mL)and diaminopimelic acid (DAP, 50 μg/ml). Colonies were selected andscreened for insertion of the knockout fragment by PCR using primersflic-3 (SEQ ID NO:234) and flic-4 (SEQ ID NO:235). pKD46 then was curedby culturing the selected kanamycin resistant strain at 42° C. andscreening for loss of ampicillin resistance. The Kanamycin resistancemarker then was cured by electroporation of a temperature sensitiveplasmid expressing the Cre recombinase (pJW1680) and Amp^(R) colonieswere selected at 30° C.; pJW168 was subsequently eliminated by growingcultures at 42° C. Selected fliC knockout clones were then tested forloss of kanamycin marker by PCR using primers flanking the sites ofdisruption (flic-3 and flic-4) and evaluation of the electrophoreticmobility on agarose gels.

Deletion of fljB

fljB was then deleted in the asd/fliC deleted YS1646 strain usingmodifications of the methods described above. Synthetic fljB genehomology arm sequences that contained 249 and 213 bases of the left handand right hand sequence, respectively, flanking the fliC gene, weresynthesized and cloned into a plasmid called pSL0148 (SEQ ID NO:231). Akanamycin gene cassette flanked by cre/loxP sites then was cloned intopSL0148 and the fljB gene knockout cassette then was PCR amplified withprimer fljb-1 (SEQ ID NO:236) and fljb-2 (SEQ ID NO:237) and gelpurified and introduced into AST-101 carrying the temperature sensitivelambda red recombination plasmid pKD46 by electroporation. The kanamycinresistance gene then was cured by cre-mediated recombination asdescribed above, and the temperature-sensitive plasmids were cured bygrowth at non-permissive temperature. The fliC and fljB gene knockoutsequences were amplified by PCR using primers flic-3 and flic-4 orfljb-3 (SEQ ID NO:238) and fljb-4 (SEQ ID NO:239), and verified by DNAsequencing. This asd⁻/fliC⁻/fljB⁻ mutant derivative of YS1646 wasdesignated AST-111.

Primer sequence information Primer SEQ name Primer sequence ID NO.flic-1 CGTTATCGGCAATCTGGAGGC 232 flic-2 CCAGCCCTTACAACAGTGGTC 233 flic-3GTCTGTCAACAACTGGTCTAACGG 234 flic-4 AGACGGTCCTCATCCAGATAAGG 235 fljb-1TTCCAGACGACAAGAGTATCGC 236 fljb-2 CCTTTAGGTTTATCCGAAGCCAGAATC 237 fljb-3CACCAGGTTTTTCACGCTGC 238 fljb-4 ACACGCATTTACGCCTGTCG 239In Vitro Characterization of Engineered S. typhimurium FlagellinKnockout Strain

The YS1646 derived asd⁻ mutant strain harboring the deletions of bothfliC and fljB, herein referred to as AST-111 or ASD/FLG, was evaluatedfor swimming motility by spotting 10 microliters of overnight culturesonto swimming plates (LB containing 0.3% agar and 50 mg/mL DAP). Whilemotility was observed for YS1646 and the asd deleted strain AST-101, nomotility was evident with the asd/fliC/fljB-deleted strain AST-111. TheAST-111 strain then was electroporated with pATIshTREX1 (a plasmidcontaining an asd gene and an shRNA targeting TREX1), to produceAST-112, and its growth rate in the absence of DAP was assessed. Asshown in FIG. 5, ASD/FLG (pATI-shTREX1) strain AST-112 was able toreplicate in LB in the absence of supplemental DAP, and grew at a ratecomparable to the asd strain containing pATIshTREX1 (AST-108). Thesedata demonstrate that the elimination of flagellin does not decrease thefitness of S. typhimurium in vitro.

Elimination of flagellin subunits decreases pyroptosis in macrophages.To demonstrate this, 5×10⁵ mouse RAW-Dual™ macrophage cells (InvivoGen,San Diego, Ca.) were infected with the asd/fliC/fljB-deleted strainharboring a low copy shTREX1 plasmid, designated AST-118, or the asddeleted strain harboring the same plasmid (AST-117) at an MOI ofapproximately 100 in a gentamycin protection assay. After 24 hours ofinfection, culture supernatants were collected and assessed for lactatedehydrogenase release as a marker of cell death using a Pierce™ LDHCytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, Ma.). AST-117induced 75% maximal LDH release, while AST-118 induced 54% maximal LDHrelease, demonstrating that the deletion of the flagellin genes reducesthe S. typhimurium-induced pyroptosis.

ASD/FLG Knockout Strain Containing shTrex1 Plasmid Demonstrates EnhancedAnti-Tumor Activity, Enhanced Interferon Gamma Responses, and IncreasedTumor Colonization in Mice Compared to Parental Asd Strain.

To assess the impact of the flagellin knockout strains administered in amurine model of colon carcinoma, 6-8 week-old female BALB/c mice (10mice per group) were inoculated SC in the right flank with CT26 (2×10⁵cells in 100 μL PBS). Mice bearing established flank tumors were IVinjected with three weekly doses of 5×10⁶ CFUs of the ASD/FLG straincontaining the pATIKan-shTREX1 plasmid (AST-113) or the ASD strain withthe same pATIKan-shTREX1 plasmid (AST-110), and compared to PBS control.Six hours following the first IV dose, mice were bled, and plasma wascollected and assessed for pro-inflammatory cytokines using the MouseInflammation Cytometric Bead Array kit and analyzed by FACS (BDBiosciences).

As shown in FIG. 6, the AST-113 strain, incapable of making flagella andcontaining the pATIshTrex1 plasmid (ASD/FLG pATI-shTREX1), demonstratedenhanced tumor control compared to the parental ASD pATI-shTREX1 strainAST-110, and significant tumor control compared to the PBS control (54%TGI, p=0.02, day 17).

Comparing the levels of systemic serum cytokines at 6 hours post IVinjection, the cytokines elicited by the AST-113 strain were comparablefor TNF-α and IL-6 as compared to the parental AST-110 strain, capableof making flagella. The levels of the potent anti-tumor immune cytokineIFN-γ were significantly higher with AST-113 compared to AST-110,indicating that the flagellin deficient strain can provide for superioranti-tumor potency over the parental asd knockout strain (FIG. 7).

At 35 days post tumor implantation (12 days after the last dose ofengineered Salmonella therapy), three mice per group were euthanized,and tumors were homogenized and plated on LB plates to enumerate thenumber of colony forming units (CFUs) per gram of tumor tissue asdescribed above. As shown in FIG. 8, the AST-113 strain, deleted of fliCand fljB and containing the pATIshTREX1 plasmid, was able to colonizetumors at least as well as the strain that only had the asd genedeletion and contained the same plasmid (AST-110). AST-113 colonizedtumors with a mean of 1.2×10⁷ CFU per gram of tissue compared with amean of 2.1×10⁶ CFU/g of tumor for AST-110, indicating that the absenceof flagellin can lead to an increased tumor colonization by greater than5 times that of strains with a functional flagella. Together, these datademonstrate that, contrary to the expectation from the art, not only isthe flagella not required for tumor colonization, but its loss canenhance tumor colonization and anti-tumor immunity.

Example 8

S. typhimurium Engineered to Express cytoLLO for Enhanced PlasmidDelivery

In this example, the asd deleted strain of YS1646 described in Example 1(AST-101) was further modified to express the listeriolysin O (LLO)protein lacking the signal sequence that accumulates in the cytoplasm ofthe Salmonella strain (referred to herein as cytoLLO). LLO is acholesterol-dependent pore-forming cytolysin that is secreted fromListeria monocytogenes and mediates phagosomal escape of bacteria. Agene encoding LLO, with codons 2-24 deleted, was synthesized with codonsoptimized for expression in Salmonella. The sequence of the open readingframe of cytoLLO is in SEQ ID NO:240. The cytoLLO gene was placed undercontrol of a promoter that induces transcription in S. typhimurium (SEQID NO: 241, reproduced below). The cytoLLO expression cassette wasinserted in single copy into the knockout-out asd locus of the asddeleted strain AST-101 using modifications of the method of Datsenko andWanner (Proc. Natl. Acad. Sci. USA (2000) 97:6640-6645), as described inExample 1.

Sequence of promoter driving expression of cytoLLO LLO promoterSEQ ID NO: 241 attatgtcttgacatgtagtgagtgggctggtataatgcagcaag

The asd deleted strain with the cytoLLO expression cassette inserted atthe asd locus (referred to herein as ASD/LLO or AST-114) was furthermodified by electroporation with a pATI plasmid encoding an asd genethat allows the strain to grow in the absence of exogenous DAP andselects for plasmid maintenance, and also contains a U6 promoter drivingexpression of shTREX1 as described in Example 6 (referred to herein asASD/LLO (pATI-shTREX1) or AST-115). As shown in FIG. 9, the ASD/LLO(pATI-shTREX1) strain AST-115 grew at a comparable rate to the asddeleted strain containing the same plasmid (pATI-shTREX1), AST-110,demonstrating that the LLO knock-in does not impact bacterial fitness invitro.

S. typhimurium Engineered to Produce cytoLLO Demonstrate PotentAnti-Tumor Activity

To determine whether the cytoLLO gene knock-in provided anti-tumorefficacy, the ASD/LLO (pATI-shTREX1) strain AST-115 was evaluated in amurine model of colon carcinoma. For this study, 6-8 week-old femaleBALB/c mice (8 mice per group) were inoculated SC in the right flankwith CT26 (2×10⁵ cells in 100 μL PBS). Mice bearing established flanktumors were IV injected with a single dose of 5×10⁶ CFUs of AST-115, andcompared to PBS control.

As shown in FIG. 10, the addition of the cytoLLO gene into the asd⁻strain ASD/LLO (pATI-shTREX1) demonstrated highly significant tumorcontrol compared to PBS control (76% TGI, p=0.002, day 28), andcomparable efficacy after a single dose to previous studies where theTREX1 shRNA plasmid containing strains were given at multiple doses.These data demonstrate the cytoLLO-mediated advantage of delivering moreplasmid into the cytosol, resulting in greater gene knockdown, therebyimproving the therapeutic efficacy of RNAi against targets such asTREX1.

Example 9 Adenosine Auxotrophic Strains of S. typhimurium

Strains provided herein are engineered to be auxotrophic for adenosine.As a result, they are attenuated in vivo because they are unable toreplicate in the low adenosine concentrations of normal tissue,therefore colonization occurs primarily in the solid tumormicroenvironment, where adenosine levels are high. The Salmonella strainYS1646 (AST-100) is a derivative of the wild type strain ATCC 14028, andwas engineered to be auxotrophic for purine due to disruption of thepurI gene (Low et al., (2004) Methods Mol. Med. 90:47-60). Subsequentanalysis of the entire genome of YS1646 demonstrated that the purI gene(synonymous with purM) was not in fact deleted, but was insteaddisrupted by a chromosomal inversion (Broadway et al. (2014) J.Biotechnol. 20:177-178), and that the entire gene is still containedwithin two parts of the YS1646 chromosome that is flanked by insertionsequences (one of which has an active transposase). The presence of thecomplete genetic sequence of the purI gene disrupted by means of achromosomal reengagement leaves open the possibility of reversion to awild type gene. While it has previously been demonstrated that purineauxotrophy of YS1646 was stable after serial passage in vitro, it wasnot clear what the reversion rate is (Clairmont et al. (2000) J. Infect.Dis. 181:1996-2002).

It is shown herein that, when provided with adenosine, YS1646 is able toreplicate in minimal medium; whereas the wild-type parental strain ATCC14028 can grow in minimal media that is not supplemented with adenosine.YS1646 was grown overnight in LB medium washed with M9 minimal mediumand diluted into M9 minimal media containing no adenosine, or increasingconcentrations of adenosine. Growth was measured using a SpectraMax® M3spectrophotometer (Molecular Devices) at 37° C., reading the OD₆₀₀ every15 minutes.

As shown in FIG. 11, strain YS1646 was able to replicate when adenosinewas provided at concentrations ranging from 11 to 300 micromolar, butwas completely unable to replicate in M9 alone or M9 supplemented with130 nanomolar adenosine. These data demonstrate that purI mutants areable to replicate in concentrations of adenosine that are found in thetumor microenvironment, but not at concentrations found in normaltissues. Engineered adenosine auxotrophic strains exemplified hereininclude strains wherein all, or portions of the purI open reading frameare deleted from the chromosome to prevent reversion to wild-type. Suchgene deletions can be achieved utilizing the lambda red system asdescribed in Example 1.

Salmonella strains containing a purI disruption, further engineered tocontain an asd gene deletion (ASD) as described in Example 1, orcontaining an asd gene deletion further engineered to have deletions offliC and fljB (ASD/FLG), as described in Example 7, or asd⁻ mutantsfurther engineered to express cytoLLO (ASD/LLO) as described in Example8, and complemented with a low copy number plasmid (pATIlow) expressingasd as described in Example 6 (Strains AST-117, AST-118, and AST-119,respectively), were also evaluated for growth in M9 minimal media. Thedata in FIG. 12 show that each strain was able to replicate whenadenosine was provided at concentrations ranging from 11 to 300micromolar, but was completely unable to replicate in M9 alone or M9supplemented with 130 nanomolar adenosine.

Example 10 Characterization and Use of the asd Gene ComplementationSystem In Vitro Growth of Strains with asd Gene Complementation

To assess fitness of the bacterial strains containing plasmids, growthcurves were performed in LB liquid media using a Spectramax plate readerat 37° C., reading the OD₆₀₀ every 15 minutes. As shown in FIG. 13,strain YS1646 containing a low copy plasmid pEQU6-shTREX1 (AST-104) grewcomparably to strain YS1646 that did not contain a plasmid (AST-100). Anasd⁻ mutant strain harboring a high copy shTREX1 plasmid with an asdgene that can complement the asd deletion (AST-110) was able toreplicate in LB in the absence of DAP, but grew slower than strainYS1646. An asd deleted strain containing an shTREX-1 expression plasmidwith a low copy number origin of replication and an asd gene that cancomplement the asd deletion (pATIlow-shTREX1), strain AST-117, grew at afaster rate than AST-110. These data demonstrate that low copy numberplasmids that complement the asd gene deletion are superior to high copynumber plasmids, as they allow for more rapid replication rates of S.typhimurium in vitro.

Intracellular Growth of asd Complemented Strains

To measure fitness of the asd⁻ mutants complemented with asd on high andlow copy plasmids, the ability of bacterial strains to replicateintracellularly in mouse tumor cell lines was assessed using agentamycin protection assay. In this assay, mouse melanoma B16.F10 cellsor mouse colon cancer CT26 cells were infected with asd⁻ mutantSalmonella strains containing plasmids that contain a complementary asdgene and have either a high copy origin of replication, AST-110 (ASDpATI-shTREX1), or a low copy origin of replication, AST-117 (ASD pATIlow copy-shTREX1). Cells were infected at a multiplicity ofapproximately 5 bacteria per cell for 30 minutes, then cells were washedwith PBS, and medium containing gentamicin was added to killextracellular bacteria. Intracellular bacteria are not killed bygentamicin, as it cannot cross the cell membrane. At various time pointsafter infection, cell monolayers were lysed by osmotic shock with waterand the cell lysates were diluted and plated on LB agar to enumeratesurviving colony forming units (CFU).

As shown in FIG. 14, the asd⁻ mutant strain complemented with a highcopy plasmid, AST-110, had an initial decline in CFUs, but was able togrow in B16.F10 cells but not in CT26 cells, demonstrating that the asdgene complementation system is sufficient to support growth insidemammalian tumor cells. The asd⁻ mutant strain containing the low copyplasmid, AST-117, was able to invade and replicate in both cell types,demonstrating that asd gene complementation on a low copy plasmid allowsfor robust asd⁻ mutant growth inside mammalian cells. The strain with alow copy plasmid replicated to higher numbers in both tumor cell types,compared to the strain with a high copy plasmid. This demonstrates thatSalmonella strains with low copy plasmids have enhanced fitness overstrains with high copy plasmids.

Plasmid Maintenance in Tumors Using Asd Complementation System

In this example, CT26 tumor-bearing mice were treated with strain YS1646containing a plasmid that expresses an shRNA targeting TREX1(pEQU6-TREX1), strain AST-104, or an asd deleted strain of YS1646containing a plasmid with a functional asd gene and an shRNA targetingTREX1 (pATI-shTREX1), strain AST-110. At 12 days after the finalSalmonella injection, tumors were homogenized, and homogenates wereserially diluted and plated on LB agar plates to enumerate the totalnumber of CFUs present, or on LB plates containing kanamycin toenumerate the number of kanamycin resistant colonies.

As shown in FIG. 15, S. typhimurium that did not have selective pressureto maintain the shRNA plasmid, AST-104, demonstrated plasmid loss, asthe percent kanamycin resistant (KanR) colonies was less than 10%. Thestrain that used the asd gene complementation system for plasmidmaintenance, AST-110, had nearly identical numbers of kanamycinresistant and kanamycin sensitive CFUs. These data demonstrate that theasd gene complementation system is sufficient to maintain the plasmid inthe context of the tumor microenvironment in mice.

Enhanced Anti-Tumor Efficacy Using Asd Complementation System

The asd complementation system is designed to prevent plasmid loss andpotentiate the anti-tumor efficacy of the inhibitory RNA delivery by S.typhimurium strains in vivo. To test this, asd deleted strainscontaining shTREX1 plasmid (AST-110) or scrambled control (AST-109) thatcontain a functional asd gene cassette were compared to YS1646containing pEQU6-shTREX1 (AST-104, a plasmid that lacks an asd genecassette and therefore does not have a mechanism for plasmidmaintenance) for anti-tumor efficacy in a murine colon carcinoma model.For this experiment, 6-8 week-old female BALB/c mice (8 mice per group)were inoculated SC in the right flank with CT26 (2×10⁵ cells in 100 μLPBS). Mice bearing established flank tumors were IV injected twice, onday 8 and day 18, with 5×10⁶ CFUs of AST-109 (ASD transformed withpATI-shScramble), AST-110 (ASD transformed with pATI-shTREX1), orAST-104 (YS1646 transformed with pEQU6-shTREX1) and compared to PBScontrol.

As shown in FIG. 16, the YS1646 strain AST-104 demonstrated tumorcontrol compared to PBS (70% TGI, day 28) despite its demonstratedplasmid loss over time. The asd⁻ strain containing the scramble controlin a pATI plasmid with the asd gene complementation system (AST-109)demonstrated tumor control compared to PBS (51% TGI, day 25), indicatingthat maintained delivery of CpG plasmids stimulates an anti-tumorresponse. The asd⁻ strain containing plasmid with the asd genecomplementation system and shTREX1 (AST-110) demonstrated the highesttumor growth inhibition compared to PBS (82% TGI, p=0.002, day 25).These data demonstrate that improved potency is achieved by preventingplasmid loss using the asd complementation system and delivery ofshTREX1, as compared to YS1646 containing plasmids without genecomplementation systems or shTREX1.

S. typhimurium Strains with Low Copy Plasmids Demonstrate SuperiorAnti-Tumor Efficacy and Tumor Colonization Compared to High CopyPlasmids

In order to compare the anti-tumor efficacy of the low copy shTREX1plasmid with the asd complementation system, relative to the high copyshTREX1 plasmid in a murine model of colon carcinoma, 6-8 week-oldfemale BALB/c mice (10 mice per group) were inoculated SC in the rightflank with CT26 (2×10⁵ cells in 100 μL PBS). Mice bearing establishedflank tumors were IV injected with two weekly doses of 5×10⁶ CFUs ofAST-117 (ASD (pATI Low-shTREX1)) or AST-110 (ASD (pATI-shTREX1) and werecompared to PBS injections as a negative control. As shown in FIG. 17,the strain with the low copy plasmid, AST-117, demonstrated superioranti-tumor efficacy compared to the strain with the high copy plasmidAST-110 (High: 59% TGI, Low: 79% TGI, p=0.042, day 25).

At the end of this tumor growth inhibition study, 4 mice from each groupwere euthanized, and tumors and spleens were homogenized as describedabove to evaluate tumor colonization and tumor to spleen colonizationratios. As shown in FIG. 18A, the strain containing the low copyplasmid, AST-117, colonized tumors at a level greater than 100 timeshigher than the strain with the high copy plasmid, AST-110. When theratio of colonies recovered from tumor and spleen were calculated,AST-117 had a greater than 10-fold higher tumor to spleen colonizationratio compared to AST-110 (FIG. 18B), demonstrating that the strain withthe low copy plasmid had greater specificity for tumor colonization thanthe strain with the high copy plasmid.

These data demonstrate a previously unknown attribute that S typhimuriumengineered to deliver plasmids encoding interfering RNAs have improvedtumor colonizing capabilities and anti-tumor efficacy when the plasmidshave low copy number origins of replication.

Example 11 Engineering of an Autolytic S. typhimurium Strain forDelivery of RNAi

As described above, the asd gene in S. typhimurium encodes aspartatesemialdehyde dehydrogenase. Deletion of this gene renders the bacteriaauxotrophic for diaminopimelic acid (DAP) when grown in vitro or invivo. This example employs an asd deletion strain (described inExample 1) that is auxotrophic for DAP and contains a plasmid suitablefor delivery of RNAi that does not contain an asd-complementing gene sothat the strain is defective for replication in vivo. This strain ispropagated in vitro in the presence of DAP and grows normally, and thenis administered as an immunotherapeutic agent to mammalian hosts whereDAP is not present, which results in autolysis of the bacteria.Autolytic strains are able to invade host cells, but are not able toreplicate due to the absence of DAP in mammalian tissues; thiscombination of attributes allows for RNAi-mediated gene knockdown andincreased safety relative to replicating strains.

In this example, the asd deleted strain of YS1646 (AST-101, described inExample 1) was further modified to express cytoLLO to generate strainAST-114 (described in Example 8), was electroporated to contain aplasmid encoding ARI-203 (a microRNA targeting TREX1), to make strainAST-120 (ASD/LLO (pEQU6-miTREX1)). When this strain is introduced intotumor bearing mice, the bacteria are taken up by host cells and enterthe Salmonella containing vacuole (SCV). In this environment, the lackof DAP prevents replication, and result in lysis of the bacteria in theSCV. Lysis of AST-120 allows for release of the plasmid, and theaccumulated cytoLLO that form pores in the cholesterol-containing SVCmembrane, resulting in efficient delivery of the plasmid into thecytosol of the host cell.

The ability of the autolytic strain AST-120, to replicate in LB in thepresence or absence of DAP was assessed using a SpectraMax® M3spectrophotometer (Molecular Devices) at 37° C., reading the OD₆₀₀ every15 minutes. As shown in FIG. 19, AST-120 is able to grow robustly in LBsupplemented with 50 μg/mL DAP, but cannot replicate in LB alone.

Increased Attenuation of Autolytic S. typhimurium in Mice To determinewhether the autolytic strain AST-120, engineered to deliver cytoLLO anda microRNA targeting TREX1, was attenuated for virulence, a medianlethal dose (LD₅₀) study was performed. Increasing doses of AST-120,ranging from 1×10⁶ to 5×10⁷ CFUs, were administered IV to C57BL/6 mice(a strain of mouse that is highly sensitive to LPS). After IVadministration, AST-120 was well tolerated at all doses with transientweight loss observed after a single dose. A second dose was administered7 days after the first dose and one mouse out of four, at the highestdose level (5×10⁷ CFUs), was found moribund and required euthanasia. Allother mice administered AST-120 experienced transient weight loss, butrecovered. These data indicate that the LD₅₀ for the autolytic strain ofS. typhimurium delivering a micro-RNA targeting TREX1 (AST-120) isgreater than 5×10⁷ CFUs. The LD₅₀ for the VNP20009 strain is known to beapproximately 5×10⁶ CFUs in C57BL/6 mice (Lee et al. (2000)International Journal of Toxicology 19:19-25), demonstrating thatAST-120 is at least 10-fold attenuated compared to VNP20009.

Antitumor Activity of Autolytic S. typhimurium

To determine whether the autolytic strain AST-120, engineered to delivercytoLLO and a microRNA targeting TREX1, was able to provide ananti-tumor response, 6-8 week-old female BALB/c mice (10 mice per group)were inoculated SC in the right flank with CT26 (2×10⁵ cells in 100 μLPBS). Mice bearing established flank tumors were IV injected with asingle dose of 5×10⁶ CFUs of the autolytic strain AST-120 (ASD/LLO(pEQU6-miTREX1)) and compared to mice treated with PBS as a control. Asshown in FIG. 20, an antitumor response was detected after only a singledose, compared to animals treated with PBS alone (52.4% TGI, p=0.02, day17). Together, these data demonstrate that S. typhimurium engineered tobe autolytic by means of DAP auxotrophy and engineered to contain aplasmid for delivery of RNAi targeting TREX1, are exquisitely attenuatedand can elicit an anti-tumor response.

Example 12 Exemplary Strains Engineered for Increased Tolerability adrAor csgD Deletion

In this example, a live attenuated strain of Salmonella typhimurium thatcontains a purI deletion, an msbB deletion, an asd gene deletion, and isengineered to deliver plasmids encoding interfering RNA, is furthermodified to delete adrA, a gene required for Salmonella typhimuriumbiofilm formation. Salmonella that cannot form biofilms are taken upmore rapidly by host phagocytic cells and are cleared more rapidly. Thisincrease in intracellular localization enhances the effectiveness ofplasmid delivery and gene knockdown by RNA interference. The increasedclearance rate from tumors/tissues increases the tolerability of thetherapy, and the lack of biofilm formation prevents colonization ofprosthetics and gall bladders in patients. In another example, a liveattenuated strain of Salmonella typhimurium that contains a purIdeletion, an msbB deletion, an asd gene deletion and is engineered todeliver plasmids encoding interfering RNA, is further modified to deletecsgD. This gene is responsible for activation of adrA, and also inducesexpression of the curli fimbriae, a TLR2 agonist. Loss of csgD alsoprevents biofilm formation, with the added benefit of inhibiting TLR2activation, thereby further reducing the bacterial virulence andenhancing delivery of RNAi.

pagP Deletion

In this example, a live attenuated strain of S. typhimurium thatcontains a purI deletion, an msbB deletion, and an asd gene deletion,and is engineered to deliver plasmids encoding interfering RNA, isfurther modified to delete pagP. The pagP gene is induced during theinfectious life cycle of S. typhimurium and encodes an enzyme thatpalmitylates lipid A. In wild type S. typhimurium, expression of pagPresults in a lipid A that is hepta-acylated. In an msbB⁻ mutant, inwhich the terminal acyl chain of the lipid A cannot be added, theexpression of pagP results in a hexa-acylated LPS. Hexa-acylated LPS hasbeen shown to be the most pro-inflammatory. In this example, a straindeleted of pagP and msbB can produce only penta-acylated LPS, allowingfor lower pro-inflammatory cytokines, enhanced tolerability, andincreased adaptive immunity when the bacteria are engineered to deliverinterfering RNAs.

hilA deletion

In this example, a live attenuated strain of Salmonella typhimurium thatcontains a purI deletion, an msbB deletion, and an asd gene deletion,and is engineered to deliver plasmids encoding interfering RNA, isfurther modified to delete hilA. hilA is a regulatory gene that isrequired for expression of the Salmonella pathogenicity island 1(SPI-1)-associated type 3 secretion system (T3SS). This secretion systemis responsible for injecting effector proteins into the cytosol ofnon-phagocytic host cells, such as epithelial cells, that cause theuptake of modified S. typhimurium. The SPI-1 T3SS has been shown to beessential for crossing the gut epithelial layer, but is dispensable forinfection when bacteria are injected parenterally. The injection of someproteins and the needle complex itself can also induce inflammasomeactivation and pyroptosis of phagocytic cells. This pro-inflammatorycell death can limit the initiation of a robust adaptive immune responseby directly inducing the death of antigen-presenting cells (APCs), aswell as modifying the cytokine milieu to prevent the generation ofmemory T-cells. In this example, the additional deletion of the hilAgene from a therapeutic Salmonella typhimurium strain that isadministered either intravenously or intratumorally focuses theSalmonella typhimurium infection towards phagocytic cells that do notrequire the SPI-1 T3SS for uptake, and then prolongs the longevity ofthese phagocytic cells. The hilA mutation reduces the quantity ofpro-inflammatory cytokines, increasing the tolerability of the therapy,as well as the quality of the adaptive immune response.

Example 13 HilA Deletion Mutants Grow Normally In Vitro

The hilA gene was deleted from the YS1646 strain of S. typhimurium withthe asd gene deleted, and the YS1646 strain deleted of asd, andflagellin genes fljB and fliC, using the lambda-derived Redrecombination system as described in Datsenko and Wanner (Proc. Natl.Acad. Sci. USA 97:6640-6645(2000)) to make the strains HilA/ASD andHilA/FLG/ASD, respectively. These strains were then electroporated witha plasmid containing a functional asd gene (to complement the deletedasd gene and to ensure plasmid maintenance in vivo) and a eukaryoticexpression cassette containing the U6 promoter driving expression of amicroRNA targeting murine TREX-1 (pATI-miTREX1). The in vitro growthrates of strains HilA/ASD (pATI-miTREX1) and HilA/FLG/ASD (pATI-miTREX1)were then determined and compared to the strains ASD (pATI-miTREX1) andYS1646 at 37° C. in LB broth, as measured by OD600 using a Spectramax 96well plate reader (Molecular devices). Each modified strain grew at arate comparable to the parental YS1646 strain in vitro, indicating thatthe hilA deletion does not reduce the fitness of the bacteria in vitro.

Example 14 HilA Deletion Mutants Induce Less Cell Death in HumanMonocytic Cells

To assess whether a hilA deletion mutant induced less pyroptosis thanstrains capable of producing SPI-1, human THP-1 monocytic cells wereinfected with a multiplicity of infection (MOI) of 1000 bacteria withstrains YS1646, ASD (pATI-miTREX1), FLG/ASD (pATI-miTREX1), and HilA/ASD(pATI-miTREX1). After 1 hour of infection, extracellular bacteria wereremoved and the media was replaced with media containing gentamycin at100 μg/mL to kill extracellular bacteria. At 4 hours post infection,cells were harvested and THP-1 cell viability was assessed usingCellTiter-Glo (Promega) reagent uptake and measuring luminescence usinga Spectramax 96 well plate reader (Molecular devices). While infectionwith YS1646 resulted in 86% cell death, infection with HilA/ASD(pATI-miTREX1) only resulted in 46% cell death. The % dead cells for ASD(pATI-miTREX1) and FLG/ASD (pATI-miTREX1) strains demonstratedintermediate phenotypes with 77% and 68% cell death, respectively. Thesedata demonstrate that hilA deleted strains induce less cell death inhuman monocytic cells than S. typhimurium strains capable of expressingSPI-1.

Example 15 HilA Deletion Mutants have Reduced Capacity to Infect HumanEpithelial Cells

HeLa cells were infected with a multiplicity of infection of 500bacteria with strains YS1646, ASD (pATI-miTREX1), FLG/ASD(pATI-miTREX1), and HilA/ASD (pATI-miTREX1). After 1 hour, extracellularbacteria were removed and the media was replaced with media containinggentamycin at 100 μg/mL to kill extracellular bacteria. At 4 hours postinfection, cells were harvested and lysed by osmotic shock, and thenumber of viable colony forming units of bacteria were enumerating byserial dilution and plating on LB agar plates. 4.6×10³ CFUs per wellwere recovered with YS1646, and only 2.0×10² CFUs were recovered withthe HilA/ASD (pATI-miTREX1) strain. Strains ASD (pATI-miTREX1) andFLG/ASD (pATI-miTREX1) demonstrated intermediate phenotypes with 8.0×10²CFUs and 6.0×10² CFUs recovered, respectively. These data demonstratethat hilA deleted strains induce less uptake in human epithelial cellsthan S. typhimurium strains capable of expressing SPI-1.

Example 16 PagP Deletion Mutants have Penta-Acylated LPS and InduceReduced Inflammatory Cytokines

The pagP gene was deleted from the asd gene-deleted strain of S.typhimurium YS1646 (which contains a purI/M and msbB deletion), usingthe lambda-derived Red recombination system as described in Datsenko andWanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)) to generate thestrain PagP/ASD. This strain was then electroporated with a plasmidcontaining a functional asd gene (to complement the deleted asd gene andto ensure plasmid maintenance in vivo) and a eukaryotic expressioncassette containing the U6 promoter driving expression of a microRNAtargeting murine TREX-1 (pATI-miTREX1) to generate the strain PagP/ASD(pATI-miTREX1). The Lipid A was then extracted from this strain andevaluated by matrix-assisted laser desorption/ionization massspectrometry (MALDI MS) and compared to wild-type S. typhimurium strainATCC 14028, strain YS1646 (which is deleted for msbB and purM), andstrain YS1646 deleted for the asd gene and complimented with thepATI-miTREX1 plasmid. Wild-type Salmonella had a minor lipid A peak witha mass of 2034, and a major peak with a mass of 1796, corresponding tothe hepta-acylated and hexa-acylated species, respectively, due to thepresence of functional msbB and purM genes. The msbB deleted strainsYS1646 and ASD (pATI-miTREX1) had major peaks at 1828 and 1585,corresponding to a mixture of hexa-acylated and penta-acylated LPS. ThemsbB and pagP deleted strain, PagP/ASD (pATI-TREX1) had only a singlepeak with a mass of 1585. These data demonstrate that deletion of pagPprevents palmitoylation of the LPS, thereby restricting it to a singlepenta-acylated species.

To determine whether the penta-acylated LPS from the pagP mutant strainsreduced TLR-4 signaling, 4 μg of purified LPS from the strains describedabove were added to THP-1 human monocytic cells, and the supernatantswere evaluated 24 hours later for the presence of inflammatory cytokinesusing a cytometric bead array (CBA) kit (BD Biosciences). The LPS fromthe PagP⁻ strain induced ¼ the amount of TNF-alpha compared to wild-typeLPS, and 7-fold less IL-6 than wild-type. The pagP⁻ mutant LPS induced22-fold less IL-6 than YS1646, demonstrating that the penta-acylated LPSspecies from a pagP⁻ mutant is significantly less inflammatory in humancells, and indicating that the pagP⁻ mutant would be better tolerated inhumans.

Example 17 FLG, HilA and PagP Deletion Mutants are More Attenuated thanStrain YS1646 in Mice

To determine whether the modified strains described above are moreattenuated than strain YS1646, a median lethal dose (LD₅₀) study wasconducted. C57BL/6 mice were injected intravenously with increasingconcentrations of strains YS1646, FLG/ASD (pATI-TREX1), HilA/ASD(pATI-TREX1), or PagP/ASD (pATI-TREX1). The LD₅₀ for strain YS1646 wasfound to be 1.6×10⁶ CFUs, which is consistent with published reports ofthis strain. The LD₅₀ for the HilA/ASD (pATI-TREX1) strain wasdetermined to be 5.3×10⁶ CFUs, demonstrating a 3-fold reduction invirulence. The LD₅₀ for the PagP/ASD (pATI-TREX1) strain was determinedto be 6.9×10⁶ CFUs, demonstrating a 4-fold reduction in virulence. TheLD₅₀ for the FLG/ASD (pATI-TREX1) strain was determined to be >7×10⁶CFUs, demonstrating a >4.4-fold reduction in virulence compared tostrain YS1646. These data indicate that the genetic modificationsdescribed above reduce the virulence of the S. typhimurium therapy andwill lead to increased tolerability in humans. In the Phase I clinicaltrial of VNP20009 (Toso et al. (2002) J. Clin. Oncol. 20(1):142-152),the presence of the bacteria in patients' tumors was only partiallyobserved at the two highest doses tested, 3E8 CFU/m² (33% presence), and1E9 CFU/m² (50% presence), indicating that the tolerable dose ofVNP20009 was too low to achieve colonization. By improving thetolerability of the strains through the modifications described above,higher doses can be administered than VNP20009. This improves both thepercentage of patients that will have their tumors colonized, and thelevel of therapeutic colonization per tumor.

Example 18 HilA Deletion Mutants Demonstrate Significant Anti-TumorActivity in Mice, and Comparable Activity to the fljB/fliC DeletionMutants

The hilA⁻ mutation should prevent upregulation of the T3SS, and preventpyroptotic cell death of infected macrophages. This should enhancetolerability and anti-tumor efficacy of the plasmid-containing targetstrains in vivo. To test this, ΔhilA/Δasd strains containing the miTREX1plasmid (HilA/ASD (pATI-miTrex1)) or vehicle control were compared toΔfljB/ΔfliC/Δasd strains containing the miTREX1 (FLG/ASD (pATI-miTrex1))plasmid for tumor efficacy in a murine colon carcinoma model. 6-8week-old female C57BL/6 mice (9 mice per group) were inoculated S.C. inthe right flank with MC38 (5×10⁵ cells in 100 μL PBS). Mice bearingestablished flank tumors were I.V. injected on day 8 with 3×10⁵ CFUs ofstrains HilA/ASD (pATI-miTrex1), FLG/ASD (pATI-miTrex1), or PBS vehiclecontrol. Body weights and tumors were measured twice weekly. Tumormeasurements were performed using electronic calipers (Fowler, Newton,Mass.). Tumor volume was calculated using the modified ellipsoid formula½(length×width²). Mice were euthanized when tumor size reached >20% ofbody weight or became necrotic, as per IACUC regulations.

The data demonstrate that the HilA/ASD (pATI-miTrex1) strain inducespotent tumor control compared to PBS (83.6% TGI, day 25), including 1/9complete tumor regressions. These data were comparable to those observedwith the FLG/ASD (pATI-miTrex1) strain compared to PBS (82.8% TGI, day25). Thus, the ΔhilA strain provides comparable or greater potency in amurine tumor model as the ΔfljB/ΔfliC strain.

Example 19 HilA Deletion Mutants Demonstrate Significantly LowerSystemic Cytokines than the Parental VNP20009 Strain, and EnhancedColonization Compared to the fljB/fliC Deletion Mutants

To test the impact of the ΔhilA/Δasd and ΔfljB/ΔfliC/Δasd strains ontumor colonization and tolerability, compared to the parental VNP20009strain, these strains, containing the miTREX1 plasmid, were evaluated ina murine colon carcinoma model. 6-8 week-old female C57BL/6 mice (3 miceper group) were inoculated S.C. in the right flank with MC38 cells(5×10⁵ cells in 100 μL PBS). Mice bearing established flank tumors wereI.V. injected on day 8 with 3×10⁵ and 1×10⁵ CFUs of HilA/ASD(pATI-miTrex1), FLG/ASD (pATI-miTrex1), VNP20009 or PBS vehicle control.Mice were bled 2 hours post-dosing, and serum assessed for systemiccytokines by a mouse inflammation cytometric bead array (BD Biosciences)on a flow cytometer (Novocyte). On day 3 post I.V. dosing, mice weresacrificed, and the tumors, spleens and livers were harvested andweighed. Tissues were homogenized in 10 mL sterile PBS (M tubes,GentleMacs, Miltenyi Biotec), then 10-fold serial dilutions wereperformed and plated on LB (Luria Broth) agar plates containingLB+kanamycin (Sigma). The following day, colony forming units (CFUs)were counted and total CFUs were assessed per gram of tissue.

Serum cytokine IL-6 levels in mice have been shown to be the mostaccurate correlate of clinical tolerability. The IL-6 levels fromVNP20009 at the 3e5 CFU dose averaged 11,755 pg/mL, compared to thebaseline 18 pg/mL of PBS control. The HilA/ASD (pATI-miTrex1) andFLG/ASD (pATI-miTrex1) IL-6 levels at the 3e5 CFU dose were bothsignificantly lower than the VNP20009 levels (3,570 pg/mL and 3,850pg/mL, respectively). These data demonstrate the significantly enhancedtolerability of the mutant strains compared to the parental VNP20009strain. Comparing the colonization of tumors, spleens and livers 3 dayspost I.V. dosing of 1e5 CFU, the overall colonization of the spleensbetween HilA/ASD (pATI-miTrex1) and FLG/ASD (pATI-miTrex1) wascomparable (HilA: 2.1e4 CFU/g, vs. FLG: 1.2e4 CFU/g), while the FLG/ASD(pATI-miTrex1) strain had somewhat lower colonization in the liver(HilA: 7.4e3 CFU/g, vs. FLG: 1.5e3 CFU/g). The tumor colonization of theHilA/ASD (pATI-miTrex1) strain was shown to be an average of 2.6e4CFU/g, which was significantly higher than the undetectable levels foundin the FLG/ASD (pATI-miTrex1) tumors at the same time point. These datademonstrate the high degree of tolerability and enhanced tumorcolonization properties of the ΔhilA/Δasd strain.

Example 20 S. typhimurium Immune Modulator Strains DemonstrateExpression of Heterologous Proteins in Human Monocytes

As described above, the hilA gene and flagellin genes fljB and fliC weredeleted from the YS1646 strain of S. typhimurium with the asd genedeleted, generating the strains HilA/ASD and FLG/ASD, respectively. Inaddition, the FLG/ASD strain was further modified to express thelisteriolysin O (LLO) protein lacking the signal sequence (cytoLLO) thataccumulates in the cytoplasm of the Salmonella strain (FLG/ASD/cLLO).These strains were electroporated with a plasmid containing anexpression cassette for the EF1c promoter and the murine cytokine IL-2(muIL-2). In addition, the FLG/ASD strain was electroporated with anexpression plasmid for IL-156 as a control for a non-cognate cytokine.Additional constructs were created using the CMV promoter.

To determine whether these strains containing expression plasmids couldinfect human monocytes and induce the production of murine IL-2, THP-1human monocytic cells were plated at 50,000 cells/well in RPMI(Corning)+10% Nu Serum (Gibco) one day prior to infection. The cellswere infected at an MOI of 50 for one hour in RPMI, then washed 3 timeswith PBS, and resuspended in RPMI+100 μg/ml Gentamycin (Sigma).Supernatants were collected 48 hours later from a 96 well plate andassessed for the concentration of murine IL-2 by ELISA (R&D Systems).The concentration of IL-2 detected in the FLG/ASD-IL156 control wellswas found to be very low as expected, and likely reflective of somecross-reactivity to endogenous human IL-2 (6.52 pg/mL). In contrast, theFLG/ASD-IL-2 strain induced an average of 35.1 pg/ml IL-2, and an evenhigher concentration of IL-2 was measured with the FLG/ASD/cLLO strain,59.8 pg/mL. The highest levels were detected in the HilA/ASD-IL-2strain, 103.4 pg/mL. These data demonstrate the feasibility ofexpressing and secreting functional heterologous proteins, such as IL-2,from the S. typhimurium immune modulator platform strains.

Example 21 Cell Infection with ΔhilA Mutant Leads to Less HumanEpithelial Cell Infection

To demonstrate that hilA deleted S. typhimurium strains are reduced intheir ability to infect epithelial cells, HeLa cervical carcinoma cellswere infected with the following S. typhimurium strains: YS1646, YS1646Δasd and YS1646 Δasd/ΔhilA, containing plasmids encoding a functionalasd gene for plasmid maintenance. 1×10⁶ HeLa cells were placed in a24-well dish with DMEM and 10% FBS. Cells were infected with log-phasecultures of S. typhimurium for 1 hour, then the cells were washed withPBS and the media was replaced with media containing 50 μg/mL gentamicinto kill extracellular bacteria. After 4 hours, the HeLa cell monolayerswere washed with PBS and lysed with 1% Triton X 100 lysis buffer torelease intracellular bacteria. The lysates were serially diluted andplated on LB agar plates to quantify the number of intracellularbacteria. The strain with the hilA deletion had a 90% reduction inrecovered CFUs compared to the strains with a functional hilA gene,demonstrating that deletion of hilA significantly decreases S.typhimurium infection of epithelial-derived cells.

Example 22 Cell Infection with ΔhilA or ΔfljB/ΔfliC Mutants Leads toLess Pyroptosis in Human Macrophages

To demonstrate that ΔhilA or ΔfljB/ΔfliC S. typhimurium strains arereduced in their ability cause cell death in macrophages, THP-1 humanmacrophage cells were infected with the following S. typhimuriumstrains: YS1646, YS1646 Δasd, YS1646 Δasd/ΔfljB/ΔfliC, and YS1646Δasd/ΔhilA, containing plasmids encoding a functional asd gene to ensureplasmid maintenance. 5×10⁴ cells were placed in a 96-well dish with DMEMand 10% FBS. Cells were infected with washed log-phase cultures of S.typhimurium for 1 hour at an MOI of 100 CFUs per cell, then the cellswere washed with PBS, and the media was replaced with media containing50 μg/mL gentamicin to kill extracellular bacteria, and 50 ng/mL ofinterferon gamma. After 24 hours, the THP-1 cells were stained withCellTiter-Glo® reagent (Promega), and the percentage of viable cells wasdetermined using a luminescent cell viability assay using a Spectramaxplate reader to quantify the luminescence. The cells infected with thehilA deletion strain had approximately 72% viable cells, whereas theYS1646-infected cells had only 38% viability, demonstrating thatdeletion of hilA prevents cell death of human macrophages. Cellsinfected with the plasmid-containing strains YS1646 Δasd and YS1646Δasd/ΔfljB/ΔfliC had 40% and 51% viability, respectively, indicatingthat the deletion of the flagellin genes also prevented cell death ofhuman macrophages.

Example 23 Infection of Human Macrophages with an Immunostimulatory S.typhimurium Strain Containing a Plasmid Encoding IL-2 ExpressionCassette Leads to Secretion of IL-2

Human THP-1 macrophages were infected with the following S. typhimuriumstrains: YS1646 Δasd/ΔfljB/ΔfliC, YS1646 Δasd-cytoLLO, and YS1646Δasd/ΔhilA, containing plasmids encoding an expression cassette formurine IL-2 under a eukaryotic promoter, and a functional asd gene toensure plasmid maintenance. 5×10⁴ cells were placed in a 96-well dishwith DMEM and 10% FBS. Cells were infected with washed log-phasecultures of S. typhimurium for 1 hour at an MOI of 50 CFUs per cell,then the cells were washed with PBS and the media was replaced withmedia containing 50 μg/mL gentamicin to kill extracellular bacteria.After 48 hours, the cellular supernatants were removed and tested formurine IL-2 using an R&D Systems™ Mouse IL-2 Quantikine ELISA Kit. Theremaining cells were stained with CellTiter-Glo® reagent (Promega), andthe percentage of viable cells was determined using a luminescent cellviability assay using a Spectramax plate reader to quantify theluminescence. The YS1646 Δasd/ΔfljB/ΔfliC, YS1646 Δasd-cytoLLO, andYS1646 Δasd/ΔhilA strains, containing plasmids encoding an expressioncassette for murine IL-2, expressed 35 pg/mL, 60 pg/mL, and 103 pg/mL ofIL-2, respectively.

Example 24 S. typhimurium Strains Expressing Murine IL-2 DemonstratePotent Tumor Growth Inhibition In Vivo

The immunostimulatory S. typhimurium strains containing deletions inhilA or the flagellin genes fljB and fliC in the YS1646 strain of S.typhimurium were combined with the asd gene deletion to form the strainsYS1646 Δasd/ΔhilA and YS1646 Δasd/ΔfljB/ΔfliC, respectively. Thesestrains were electroporated with a plasmid containing an expressioncassette for the EF1c promoter and the murine cytokine IL-2.

To show that the S. typhimurium strains containing the IL-2 expressionplasmids induce anti-tumor efficacy, the Δasd/ΔhilA strains containingthe muIL-2 plasmid, or the Δasd/ΔfljB/ΔfliC strains containing themuIL-2 plasmid, were compared to vehicle control. 6-8 week-old femaleC57BL/6 mice (5 mice per group) were inoculated S.C. in the right flankwith MC38 cells (5×10⁵ cells in 100 μL PBS). Mice bearing establishedflank tumors were IV injected on day 8 with 5×10⁵ CFUs of Δasd/ΔhilA(pATI-muIL-2), Δasd/ΔfljB/ΔfliC (pATI-muIL-2), or PBS vehicle control.Body weights and tumors were measured twice weekly. Tumor measurementswere performed using electronic calipers (Fowler, Newton, Mass.). Tumorvolume was calculated using the modified ellipsoid formula,½(length×width²). Mice were euthanized when tumor size reached >20% ofbody weight or became necrotic, as per IACUC regulations.

The experiment demonstrated that the Δasd/ΔhilA (pATI-muIL-2) strainelicited significant tumor control compared to PBS (P=0.003, day 21).These data were comparable to that observed with the Δasd/ΔfljB/ΔfliC(pATI-muIL-2) strain, which also demonstrated significant tumor growthinhibition compared to PBS (P=0.005, day 21). Thus, both strainsdemonstrate the ability of expressed IL-2 to potently inhibit tumorgrowth inhibition in a model of colorectal carcinoma.

Example 25 pagP⁻, fljB⁻/fliC⁻, and pagP⁻/fljB⁻/fliC⁻ Strains DemonstrateSignificantly Higher Viability in Human Serum Compared to VNP20009(YS1646)

As described herein, VNP20009 (YS1646) exhibits limited tumorcolonization in humans after systemic administration. It is shown hereinthat VNP20009 is inactivated by complement factors in human blood. Todemonstrate this, strains YS1646 and E. coli D10B were compared toexemplary immunostimulatory bacteria provided herein that containadditional mutations that alter the surface of the bacteria. Thesestrains were YS1646 (pagP⁻), YS1646 (fljB⁻/fliC⁻), and YS1646(pagP⁻/fljB⁻/fliC⁻). These three strains, in addition to YS1646 and E.coli D10B cultures, were incubated with serum or heat-inactivated (HI)serum from either pooled mouse blood or pooled healthy human donors(n=3), for 3 hours at 37° C. After incubation with serum, bacteria wereserially diluted and plated on LB agar plates, and the colony formingunits (CFUs) were measured.

In mouse serum, all strains remained 100% viable and were completelyresistant to complement inactivation. In human serum, all strains were100% viable in the heat-inactivated serum. The E. coli D10B strain wascompletely eliminated after 3 hours in whole human serum. The YS1646strain exhibited only 6.37% of live colonies, demonstrating that tumorcolonization of the YS1646 clinical strain was limited due to complementinactivation in human blood. For the YS1646 (fljB⁻/fliC⁻) strain, 31.47%of live colonies remained, and for the YS1646 (pagP⁻) strain, 72.9% oflive colonies remained, after incubation with human serum for 3 hours.The combined YS1646 (pagP⁻/fljB⁻/fliC⁻) strain was completely resistantto complement in human serum.

These data explain why VNP20009 had very low tumor colonization whensystemically administered. It is shown herein that VNP20009 (YS1646) ishighly sensitive to complement inactivation in human serum, but notmouse serum. These data explain why limited tumor colonization wasobserved in humans, while mouse tumors were colonized at a high level.The fljB/fliC or pagP deletions, or the combination of these mutations,partially or completely rescues this phenotype. Thus, the enhancedstability observed in human serum with the fljB/fliC, pagP, orpagP/fljB/fliC deletion strains provides for increased human tumorcolonization.

These data and other provided herein (see, e.g., Examples 7, 16 and 17,above) show that deletion of the flagella and/or pagP increases tumorcolonization, improves tolerability, and increases the anti-tumoractivity of the immunostimulatory bacteria. Example 16 demonstrates thatLPS from immunostimulatory bacteria that are pagP⁻ induced 22-fold lessIL-6 than LPS from YS1646, and therefore pagP⁻ bacteria are lessinflammatory in human cells. Example 17 demonstrates that each and allof FLG, HilA and PagP deletion mutants are more attenuated than YS1646.

Immunostimulatory bacteria, such as Salmonella strains, includingwild-type strains, that are one or both of flagellin and pagP⁻ exhibitproperties that increase tumor/tumor microenvironment colonization andincrease anti-tumor activity. Such strains can be used to deliver atherapeutic payload, such as an immunotherapeutic product and/or otheranti-tumor product, and also can include modifications that improvetherapeutic properties, such as deletion of hilA, and/or msbB, adenosineauxotrophy, and other properties as described elsewhere herein. Theresulting strains are more effectively targeted to the tumor/tumormicroenvironment, by virtue of the modifications that alter infectivity,toxicity to certain cells, and nutritional requirements, such asauxotrophy for purines, that are provided in the tumor environment.

Example 26 fljB⁻/fliC⁻ Immunostimulatory Bacterial Strain DemonstratesTumor Myeloid Cell-Specific Colonization In Vivo

The asd and flagellin (fljB/fliC) genes were deleted from strain YS1646,which is purI⁻/msbB⁻, using the lambda-derived Red recombination systemas described previously (see, Datsenko and Wanner (2000) Proc. Natl.Acad. Sci. USA 97:6640-6645), to generate the strain YS1646 ΔFLG/ΔASD.Strain YS1646 ΔFLG/ΔASD was then transformed by electroporation with thebacterial plasmid pRPSM-mCherry, containing 1) a functional asdexpression cassette to complement the chromosomal deletion of asd for invivo plasmid maintenance, and 2) a constitutive mCherry expressioncassette under control of the bacterial rpsm promoter (rpsm-mCherry).Bacterial colonies transformed with this plasmid were visibly red incolor, due to expression of the mCherry red fluorescent protein. Toevaluate tumor colonization, the transformed bacterial strain (YS1646ΔFLG/ΔASD (pRPSM-mCherry)) was tested in vivo in a murine coloncarcinoma model. 6-8 week-old female C57BL/6 mice (3 mice per group)were inoculated subcutaneously in the right flank with MC38 cells (5×10⁵cells in 100 μL PBS). Mice bearing large, established flank tumors wereintravenously injected with 1×10⁶ CFUs of YS1646 ΔFLG/ΔASD(pRPSM-mCherry). Tumors were harvested 3 days later and dissociated intoa single cell suspension (Miltenyi Biotec). Cells were stained withZombie Aqua™ fixable viability dye (BioLegend), which penetrates dead,but not live, cells. The cells were incubated with the followingantibodies: Brilliant Violet 510™ anti-mouse CD45 (clone 30-F11,BioLegend); Brilliant Violet 421™ anti-mouse CD8a (clone 53-6.7,BioLegend); PE anti-mouse CD3ε (clone 145-2C11, BioLegend); FITCanti-mouse CD4 (clone RM4-5, BioLegend); PE/Cy7 anti-mouse/human CD11b(clone M1/70, BioLegend); Brilliant Violet 785™ anti-mouse Ly6C (cloneHK1.4, BioLegend); Brilliant Violet 605™ anti-mouse Ly6G (clone 1A8,BioLegend); APC anti-mouse F4/80 (clone BM8, BioLegend); and PercP/Cy5.5anti-mouse CD24 (clone M1/69, Biolegend). The cells were then sorted byflow cytometry (Novocyte) using the various surface markers and mCherry⁺(PE Texas Red), to determine/localize bacterial uptake by the harvestedcells.

CD45⁻ cells, which include stromal and tumor cells, demonstrated nodetectable bacterial colonization, with 0.076% cells being positive formCherry, compared to a background staining level of 0.067%. CD45⁺tumor-infiltrating myeloid cells were positive for mCherry, with 7.27%of monocytes, 3.33% of dendritic cells (DCs), and 8.96% of macrophagesbeing positive for mCherry, indicating uptake of the YS1646 ΔFLG/ΔASD(pRPSM-mCherry) bacteria. A control strain, containing intact flagella,was tested in parallel. Unlike the ΔFLG strain, the flagellin⁺ controlstrain infected CD45⁻ cells, with 0.36% of CD45⁻ cells being positivefor mCherry, which was 5.37-fold greater than background staining(0.067%). The flagellin⁺ control strain also infected CD45⁺ myeloidpopulations, with 5.71% of monocytes, 5.56% of DCs, and 9.52% ofmacrophages being positive for mCherry. These data indicate thatflagella knockout strains accumulate in the myeloid cell populations ofthe tumor, but not in the tumor or stromal cells, whereas strains withintact flagella infect all cell types. Thus, flagella knockout strainsdemonstrate tumor myeloid-specific colonization in vivo.

Example 27 Flagella Knockout (ΔfljB/ΔfliC) and ΔpagP Strains DemonstrateIncreased Tolerability and Decreased Immunogenicity In Vivo

The pagP gene was deleted from the S. typhimurium strains YS1646 ΔASDand YS1646 ΔFLG/ΔASD, generating the strains YS1646 ΔPagP/ΔASD andYS1646 ΔPagP/ΔFLG/ΔASD, respectively. Strains YS1646 ΔFLG/ΔASD, YS1646ΔPagP/ΔASD, and YS1646 ΔPagP/ΔFLG/ΔASD were transformed byelectroporation with plasmids encoding the asd gene, as well as aeukaryotic expression cassette encoding murine IL-2 (muIL-2). To testthe tolerability of these strains in vivo, an LD₅₀ study was performedin 6-8 week old female BALB/c mice. The mice were intravenously injectedwith 3×10⁵, 1×10⁶, 3×10⁶, 1×10⁷, or 3×10⁷ CFUs of strains YS1646, YS1646ΔFLG/ΔASD (muIL-2), YS1646 ΔPagP/ΔASD (muIL-2), or YS1646ΔPagP/ΔFLG/ΔASD (muIL-2). The mice were then monitored for morbidity andmortality, and the LD₅₀ values were calculated. The results are shown inthe table below.

Bacterial Strain LD₅₀ (CFUs) YS1646 7.24 × 10⁶ YS1646 ΔFLG/ΔASD (muIL-2)2.07 × 10⁷ YS1646 ΔPagP/ΔASD (muIL-2) 1.39 × 10⁷ YS1646 ΔPagP/ΔFLG/ΔASD(muIL-2) Not calculated

The LD₅₀ values for the YS1646 ΔFLG/ΔASD (muIL-2) and YS1646 ΔPagP/ΔASD(muIL-2) strains were higher than the LD₅₀ value for the parental YS1646strain, indicating that the tolerability of the flagellin⁻ and pagPdeletion mutants, expressing murine IL-2, was higher in vivo. The LD₅₀for strain YS1646 ΔPagP/ΔFLG/ΔASD (muIL-2) was not calculated, as noanimals died during the duration of the study, but was greater than6.2×10⁷ CFUs, representing a near 10-fold improvement in thetolerability, compared to the parental YS1646 strain.

To compare the immunogenicity of the different bacterial strains, micethat survived the 3×10⁶ CFU dose (N=5, except YS1646, where N=4) werebled at day 40 post intravenous dosing, and anti-Salmonella serumantibodies were titered. Sera from mice treated with the various mutantbacterial strains, and from control mice, were seeded in a 96-well PCRplate and serially diluted in PBS. Cultures of the S. typhimuriumstrains containing the pRPSM-mCherry plasmid were spun down and washed,then resuspended in flow-cytometry fixation buffer. For the assay, 25 μlof the mCherry⁺ bacterial cultures, containing 1×10⁶ CFUs, were added tothe sera and incubated for 25 minutes at room temperature. Followingincubation, the bacterial samples were centrifuged and washed twice withPBS by spinning them at 4000 RPM for 5 min, and then resuspended in PBScontaining a secondary goat anti-mouse Fc Alexa Fluor® 488 antibody(1/400 dilution from stock), and incubated for 25 minutes at roomtemperature in the dark. The samples were then washed three times withPBS by spinning them at 4000 RPM for 5 min, resuspended in PBS, andanalyzed by flow cytometry (Novocyte). The results showed that the miceinjected with parental strain YS1646 had the highest serum antibodytiters, with an average mean fluorescence intensity (MFI) of29,196±20,730. Sera from mice injected with strain YS1646 ΔFLG/ΔASD(muIL-2) had an MFI of 7,941±9,290; sera from mice injected with strainYS1646 ΔPagP/ΔASD (muIL-2) had an MFI of 3,454±3,860; and sera from miceinjected with strain YS1646 ΔPagP/ΔFLG/ΔASD (muIL-2), had the lowestserum antibody titers, with an MFI of 2,295±2,444. The data demonstratethat deletion of the genes encoding the flagella (fljB/fliC) or pagPresult in strains with decreased immunogenicity, and that thecombination of mutations (ΔPagP/ΔFLG) further decreases theimmunogenicity, compared to the parental strain without the deletions.

Overall, the data demonstrate the improved tolerability and decreasedimmunogenicity of the ΔFLG and ΔPagP strains, with the ΔPagP/ΔFLG/ΔASDstrain demonstrating the most favorable tolerability and lowestimmunogenicity.

Since modifications will be apparent to those of skill in the art, it isintended that this invention be limited only by the scope of theappended claims.

What is claimed:
 1. An immunostimulatory bacterium, comprising a plasmidcontaining a sequence of nucleotides encoding an anti-cancer therapeuticproduct under control of a eukaryotic promoter, wherein: the genome ofthe immunostimulatory bacterium is modified so that the bacteriumpreferentially infects tumor-resident immune cells, and/or is modifiedto increase colonization of tumors, and/or the genome of theimmunostimulatory bacterium is modified so that it induces less celldeath in tumor-resident immune cells (decreases pyroptosis), whereby theimmunostimulatory bacterium accumulates in tumors or in the tumormicroenvironment or in tumor-resident immune cells to thereby deliverthe encoded therapeutic product; and the anti-cancer therapeutic productis expressed under the control of eukaryotic regulatory sequences,whereby the product is expressed in a eukaryotic host.
 2. Theimmunostimulatory bacterium of claim 1 that is modified to have reducedpathogenicity, whereby infection of epithelial and/or other non-immunecells is reduced relative to the bacterium without the modification. 3.The immunostimulatory bacterium of claim 1, wherein the genome of theimmunostimulatory bacterium is modified so that it induces less celldeath in tumor-resident immune cells.
 4. The immunostimulatory bacteriumof claim 1, wherein the genome of the immunostimulatory bacterium ismodified whereby the bacterium is flagellin⁻ (fliC⁻/fljB⁻) and/or pagP⁻,wherein the wild-type bacterium comprises flagella.
 5. Theimmunostimulatory bacterium of claim 4 that is flagellin⁻ (fliC⁻/fljB⁻).6. The immunostimulatory bacterium of claim 5, wherein the bacterium isauxotrophic for adenosine, or for adenosine and adenine.
 7. Theimmunostimulatory bacterium of claim 4, wherein the bacterium isflagellin⁻ (fliC⁻/fljB⁻) and pagP⁻.
 8. The immunostimulatory bacteriumof claim 1, wherein the genome of the immunostimulatory bacterium ismodified whereby the bacterium is pagP⁻/msbB⁻ or is pagP⁻.
 9. Theimmunostimulatory bacterium of claim 1, wherein the immunostimulatorybacterium is flagellin⁻ (fliC⁻/fljB⁻) and one or more of purI⁻ (purM⁻),msbB⁻, purD⁻, pagP⁻, adrA⁻, csgD⁻, qseC⁻, and hilA⁻.
 10. Theimmunostimulatory bacterium of claim 4, wherein the immunostimulatorybacterium also is msbB⁻/asd⁻/purI⁻.
 11. The immunostimulatory bacteriumof claim 1, wherein the immunostimulatory bacterium is hilA⁻ and/orflagellin⁻ (fliC⁻/fljB⁻).
 12. The immunostimulatory bacterium of claim11, wherein the immunostimulatory bacterium is hilA⁻, pagP⁻, andflagellin⁻ (fliC⁻/fljB⁻).
 13. The immunostimulatory bacterium of claim4, wherein the immunostimulatory bacterium is aspartate-semialdehydedehydrogenase⁻ (asd⁻).
 14. The immunostimulatory bacterium of claim 1,wherein the immunostimulatory bacterium is auxotrophic for adenosine, orfor adenosine and adenine.
 15. The immunostimulatory bacterium of claim1, wherein the therapeutic product is an immunostimulatory protein that,upon expression, confers or contributes to anti-tumor immunity in thetumor microenvironment.
 16. The immunostimulatory bacterium of claim 15,wherein the immunostimulatory protein that confers or contributes toanti-tumor immunity in the tumor microenvironment is a cytokine or achemokine.
 17. The immunostimulatory bacterium of claim 15, wherein theimmunostimulatory protein that confers or contributes to anti-tumorimmunity in the tumor microenvironment is selected from among one ormore of: IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-2 that hasattenuated binding to IL-2Ra, IL-15/IL-15R alpha chain complex, IL-18,IL-21, IL-23, IL-36γ, IL-2 modified so that it does not bind to IL-2Ra,CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ, CCL3,CCL4, CCL5, proteins that are involved in or that effect or potentiaterecruitment/persistence of T cells, CD40, CD40 ligand (CD40L), CD28,OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of theB7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, andmembers of the tumor necrosis factor receptor (TNFR) superfamily. 18.The immunostimulatory bacterium of claim 1, wherein the therapeuticproduct is an antibody or antigen-binding fragment thereof.
 19. Theimmunostimulatory bacterium of claim 18, wherein the antibody orantigen-binding fragment thereof is an antagonist of PD-1, PD-L1,CTLA-4, VEGF, VEGFR2, CD47, or IL-6.
 20. The immunostimulatory bacteriumof claim 1, wherein the nucleic acid encoding the therapeutic product isoperatively linked for expression to a nucleic acid encoding a secretorysignal, whereby, upon expression in a host, the therapeutic product issecreted.
 21. The immunostimulatory bacterium of claim 2 that is asd⁻,purI⁻, msbB⁻, and one or both of flagellin⁻ (fliC⁻/fljB⁻) and pagP⁻. 22.The immunostimulatory bacterium of claim 1, wherein the plasmid ispresent in low copy number or medium copy number.
 23. Theimmunostimulatory bacterium of claim 1, wherein the plasmid is presentin low copy number.
 24. The immunostimulatory bacterium of claim 1,wherein: the therapeutic product is an immunostimulatory protein; theimmunostimulatory protein, when expressed in a mammalian subject,confers or contributes to anti-tumor immunity in the tumormicroenvironment; the immunostimulatory protein is encoded on a plasmidin the bacterium under control of a eukaryotic promoter; and the genomeof the immunostimulatory bacterium is modified so that it preferentiallyinfects tumor-resident immune cells.
 25. The immunostimulatory bacteriumof claim 1, comprising a plasmid encoding a therapeutic product,wherein: the immunostimulatory bacterium is a Salmonella species; theimmunostimulatory bacterium is an adenosine auxotroph, and is flagellin⁻(fliC⁻/fljB⁻); and the therapeutic product encoded on the plasmid isexpressed under the control of eukaryotic regulatory sequences.
 26. Theimmunostimulatory bacterium of claim 1, wherein the therapeutic productis expressed under control of an RNA polymerase II promoter that is aviral promoter or a mammalian RNA polymerase II promoter.
 27. Theimmunostimulatory bacterium of claim 26, wherein the promoter isselected from among a cytomegalovirus (CMV) promoter, an SV40 promoter,an Epstein Barr virus (EBV) promoter, a herpes virus promoter, anadenovirus promoter, an elongation factor-1 alpha (EF-1 alpha) promoter,a UBC promoter, a PGK promoter, a CAGG promoter, an EIF4A1 promoter, aCBA promoter (chicken beta actin), an MND promoter, a CD68 promoter, aCAG promoter, and a GAPDH promoter.
 28. The immunostimulatory bacteriumof claim 25, wherein the regulatory sequences comprise a terminatorand/or promoters selected from among SV40, hGH, BGH, chickenbeta-globulin, and rbGlob (rabbit globulin) genes.
 29. Theimmunostimulatory bacterium of claim 4, wherein the nucleic acidencoding the therapeutic product on the plasmid is operatively linked tonucleic acid encoding a secretory signal, whereby, upon expression in ahost, the product is secreted.
 30. The immunostimulatory bacterium ofclaim 1 that has a modification in the type 3 secretion system or type 4secretion system.
 31. The immunostimulatory bacterium of claim 1,wherein the therapeutic product is a cytotoxin.
 32. Theimmunostimulatory bacterium of claim 1, wherein the therapeutic productis an anti-tumor antibody or antigen-binding fragment thereof or asingle chain antibody.
 33. The immunostimulatory bacterium of claim 32,wherein the antibody is an anti-PD-1, anti-CTLA4, anti-PD-L1, anti-VEGF,anti-VEGFR2, anti-IL-6, or anti-CD47 antibody, or an antigen-bindingfragment thereof.
 34. The immunostimulatory bacterium of claim 1,wherein the therapeutic product is a tumor antigen or a tumorneoantigen.
 35. The immunostimulatory bacterium of claim 1, wherein thebacterium is a strain of Salmonella, Shigella, E. coli, Bifidobacteriae,Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria,Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter,Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella,Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, oran attenuated strain thereof or modified strain thereof of any of thepreceding list of bacterial strains.
 36. The immunostimulatory bacteriumof claim 1 that is a strain of Salmonella.
 37. The immunostimulatorybacterium of claim 36 that is a Salmonella typhimurium strain.
 38. Theimmunostimulatory bacterium of claim 37, wherein the immunostimulatorybacterium is derived from a Salmonella typhimurium strain selected fromamong strains designated as AST-100, VNP20009, YS1646 (ATCC #202165),RE88, SL7207, χ 8429, χ 8431, or χ 8468, or wild-type strain ATCC 14028.39. The immunostimulatory bacterium of claim 1 that comprises nucleicacid encoding two therapeutic products, wherein different promoters andterminators control expression of each therapeutic product.
 40. Theimmunostimulatory bacterium of claim 1, wherein the plasmid that encodesthe therapeutic product comprises a construct that includes one or moreof an enhancer, a promoter, an IRES, the open reading frame encoding thetherapeutic product, and a polyA tail.
 41. A pharmaceutical composition,comprising the immunostimulatory bacterium of claim 1 in apharmaceutically acceptable vehicle.
 42. The pharmaceutical compositionof claim 41 that is formulated for parenteral administration, or forintratumoral administration, or for intra-peritoneal administration. 43.The pharmaceutical composition of claim 41 that is formulated forsystemic administration.
 44. A method of treatment of cancer thatcomprises a solid tumor or a hematological malignancy in a subject,comprising administering the pharmaceutical composition of claim
 41. 45.The method of claim 44, wherein the pharmaceutical composition isadministered systemically.
 46. The method of claim 44, wherein thepharmaceutical composition is administered intra-tumorally or isadministered intra-peritoneally.
 47. The method of claim 44, wherein thesubject is a human.
 48. The method of claim 44, wherein the cancercomprises a solid tumor.
 49. The method of claim 44, wherein thetreatment comprises combination therapy in which a second anti-canceragent or treatment is administered.
 50. The method of claim 49, whereinthe second anti-cancer agent or treatment is an immunotherapy.
 51. Themethod of claim 49, wherein the second anti-cancer agent or treatmentcomprises oncolytic virus therapy, or immunotherapy to inhibit an immunecheckpoint.
 52. The method of claim 51, wherein the second anti-canceragent or treatment is an immunotherapy that comprises administration ofan anti-PD-1, or anti-PD-L1, or anti-CTLA4, or anti-IL6, or anti-VEGF,or anti-VEGFR2, or anti-CD47 antibody, or an antigen-binding fragmentthereof.
 53. The method of claim 44, wherein the cancer is selected fromamong leukemia, lymphoma, gastric cancer, and cancer of the breast,heart, lung, bile duct, small intestine, colon, spleen, kidney, bladder,head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone,bone marrow, blood, thymus, uterus, testicles, cervix, and liver. 54.The method of claim 44, wherein the immunostimulatory bacterium is aSalmonella species.
 55. The method of claim 54, wherein the bacterium isa Salmonella typhimurium strain.
 56. The method of claim 55, wherein theSalmonella typhimurium is derived from a wild-type Salmonellatyphimurium strain having all of the identifying characteristics of thestrain deposited under ATCC accession no. 14028 or is the straindeposited under ATCC accession no.
 14028. 57. An isolated cell orcultured cells, comprising the immunostimulatory bacterium of claim 1.58. The cell of claim 57 that is an immune cell, a stem cell, a tumorcell, or a primary cell line.
 59. The cell of claim 57 that is a T-cell.60. The cell of claim 57 that is produced ex vivo by infecting the cellwith the immunostimulatory bacterium.
 61. A method of treatment ofcancer, comprising administering the cell or cultured cells of claim 57to a subject with a cancer that comprises a solid tumor or is ahematological malignancy.